Compositions and methods for the treatment of immune related diseases

ABSTRACT

The present invention relates to compositions containing novel proteins and methods of using those compositions for the diagnosis and treatment of immune related diseases.

FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods usefulfor the diagnosis and treatment of immune related diseases.

BACKGROUND OF THE INVENTION

[0002] Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

[0003] Though the genesis of these diseases often involves multisteppathways and often multiple different biological systems/pathways,intervention at critical points in one or more of these pathways canhave an ameliorative or therapeutic effect. Therapeutic intervention canoccur by either antagonism of a detrimental process/pathway orstimulation of a beneficial process/pathway.

[0004] Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases,non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, etc.

[0005] T lymphocytes (T cells) are an important component of a mammalianimmune response. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen -MHC complex on an antigenpresenting cell. Helper T cells also secrete a variety of cytokines,i.e., lymphokines, which play a central role in the activation of Bcells, cytotoxic T cells and a variety of other cells which participatein the immune response.

[0006] A central event in both humoral and cell mediated immuneresponses is the activation and clonal expansion of helper T cells.Helper T cell activation is initiated by the interaction of the T cellreceptor (TCR)—CD3 complex with an antigen-MHC on the surface of anantigen presenting cell. This interaction mediates a cascade ofbiochemical events that induce the resting helper T cell to enter a cellcycle (the G0 to G1 transition) and results in the expression of a highaffinity receptor for IL-2 and sometimes IL-4. The activated T cellprogresses through the cycle proliferating and differentiating intomemory cells or effector cells.

[0007] In addition to the signals mediated through the TCR, activationof T cells involves additional costimulation induced by cytokinesreleased by the antigen presenting cell or through interactions withmembrane bound molecules on the antigen presenting cell and the T cell.The cytokines IL-1 and IL-6 have been shown to provide a costimulatorysignal. Also, the interaction between the B7 molecule expressed on thesurface of an antigen presenting cell and CD28 and CTLA-4 moleculesexpressed on the T cell surface effect T cell activation. Activated Tcells express an increased number of cellular adhesion molecules, suchas ICAM-1, integrins, VLA4, LFA-1, CD56, etc.

[0008] T-cell proliferation in a mixed lymphocyte culture or mixedlymphocyte reaction (MLR) is an established indication of the ability ofa compound to stimulate the immune system. In many immune responses,inflammatory cells infiltrate the site of injury or infection. Themigrating cells may be neutrophilic, eosinophilic, monocytic orlymphocytic as can be determined by histologic examination of theaffected tissues. Current Protocols in Immunology, ed. John E. Coligan,1994, John Wiley & Sons, Inc.

[0009] Immune related diseases could be treated by suppressing theimmune response. Using neutralizing antibodies that inhibit moleculeshaving immune stimulatory activity would be beneficial in the treatmentof immune-mediated and inflammatory diseases. Molecules which inhibitthe immune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

SUMMARY OF THE INVENTION

[0010] A. Embodiments

[0011] The present invention concerns compositions and methods usefulfor the diagnosis and treatment of immune related disease in mammals,including humans. The present invention is based on the identificationof proteins (including agonist and antagonist antibodies) which eitherstimulate or inhibit the immune response in mammals. Immune relateddiseases can be treated by suppressing or enhancing the immune response.Molecules that enhance the immune response stimulate or potentiate theimmune response to an antigen. Molecules which stimulate the immuneresponse can be used therapeutically where enhancement of the immuneresponse would be beneficial. Alternatively, molecules that suppress theimmune response attenuate or reduce the immune response to an antigen(e.g., neutralizing antibodies) can be used therapeutically whereattenuation of the immune response would be beneficial (e.g.,inflammation). Accordingly, the PRO polypeptides, agonists andantagonists thereof are also useful to prepare medicines and medicamentsfor the treatment of immune-related and inflammatory diseases. In aspecific aspect, such medicines and medicaments comprise atherapeutically effective amount of a PRO polypeptide, agonist orantagonist thereof with a pharmaceutically acceptable carrier.Preferably, the admixture is sterile.

[0012] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisescontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native sequence PRO polypeptide. In a specificaspect, the PRO agonist or antagonist is an anti-PRO antibody.

[0013] In another embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide or an agonist or antagonist antibodywhich binds the polypeptide in admixture with a carrier or excipient. Inone aspect, the composition comprises a therapeutically effective amountof the polypeptide or antibody. In another aspect, when the compositioncomprises an immune stimulating molecule, the composition is useful for:(a) increasing infiltration of inflammatory cells into a tissue of amammal in need thereof, (b) stimulating or enhancing an immune responsein a mammal in need thereof, (c) increasing the proliferation ofT-lymphocytes in a mammal in need thereof in response to an antigen, (d)stimulating the activity of T-lymphocytes or (e) increasing the vascularpermeability. In a further aspect, when the composition comprises animmune inhibiting molecule, the composition is useful for: (a)decreasing infiltration of inflammatory cells into a tissue of a mammalin need thereof, (b) inhibiting or reducing an immune response in amammal in need thereof, (c) decreasing the activity of T-lymphocytes or(d) decreasing the proliferation of T-lymphocytes in a mammal in needthereof in response to an antigen. In another aspect, the compositioncomprises a further active ingredient, which may, for example, be afurther antibody or a cytotoxic or chemotherapeutic agent. Preferably,the composition is sterile.

[0014] In another embodiment, the invention concerns a method oftreating an immune related disorder in a mammal in need thereof,comprising administering to the mammal an effective amount of a PROpolypeptide, an agonist thereof, or an antagonist thereto. In apreferred aspect, the immune related disorder is selected form the groupconsisting of: systemic lupus erythematosis, rheumatoid arthritis,osteoarthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis, idiopathic inflammatory myopathies, Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, thyroiditis, diabetes mellitus,immune-mediated renal disease, demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious, autoimmune chronic active hepatitis, primary biliarycirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple'sdisease, autoimmune or immune-mediated skin diseases including bullousskin diseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases such as asthma, allergic rhinitis, atopic dermatitis,food hypersensitivity and urticaria, immunologic diseases of the lungsuch as eosinophilic pneumonias, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease.

[0015] In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody. In one aspect, the presentinvention concerns an isolated antibody which binds a PRO polypeptide.In another aspect, the antibody mimics the activity of a PRO polypeptide(an agonist antibody) or conversely the antibody inhibits or neutralizesthe activity of a PRO polypeptide (an antagonist antibody). In anotheraspect, the antibody is a monoclonal antibody, which preferably hasnonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a monoclonal antibody, a single-chain antibody, or ananti-idiotypic antibody.

[0016] In yet another embodiment, the present invention provides acomposition comprising an anti-PRO antibody in admixture with apharmaceutically acceptable carrier. In one aspect, the compositioncomprises a therapeutically effective amount of the antibody.Preferably, the composition is sterile. The composition may beadministered in the form of a liquid pharmaceutical formulation, whichmay be preserved to achieve extended storage stability. Alternatively,the antibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

[0017] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0018] (a) a composition of matter comprising a PRO polypeptide oragonist or antagonist thereof;

[0019] (b) a container containing said composition; and

[0020] (c) a label affixed to said container, or a package insertincluded in said container referring to the use of said PRO polypeptideor agonist or antagonist thereof in the treatment of an immune relateddisease. The composition may comprise a therapeutically effective amountof the PRO polypeptide or the agonist or antagonist thereof.

[0021] In yet another embodiment, the present invention concerns amethod of diagnosing an immune related disease in a mammal, comprisingdetecting the level of expression of a gene encoding a PRO polypeptide(a) in a test sample of tissue cells obtained from the mammal, and (b)in a control sample of known normal tissue cells of the same cell type,wherein a higher or lower expression level in the test sample ascompared to the control sample indicates the presence of immune relateddisease in the mammal from which the test tissue cells were obtained.

[0022] In another embodiment, the present invention concerns a method ofdiagnosing an immune disease in a mammal, comprising (a) contacting ananti-PRO antibody with a test sample of tissue cells obtained from themammal, and (b) detecting the formation of a complex between theantibody and a PRO polypeptide, in the test sample; wherein theformation of said complex is indicative of the presence or absence ofsaid disease. The detection may be qualitative or quantitative, and maybe performed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger quantity of complexes formed in the test sample indicates thepresence or absence of an immune disease in the mammal from which thetest tissue cells were obtained. The antibody preferably carries adetectable label. Complex formation can be monitored, for example, bylight microscopy, flow cytometry, fluorimetry, or other techniques knownin the art. The test sample is usually obtained from an individualsuspected of having a deficiency or abnormality of the immune system.

[0023] In another embodiment, the invention provides a method fordetermining the presence of a PRO polypeptide in a sample comprisingexposing a test sample of cells suspected of containing the PROpolypeptide to an anti-PRO antibody and determining the binding of saidantibody to said cell sample. In a specific aspect, the sample comprisesa cell suspected of containing the PRO polypeptide and the antibodybinds to the cell. The antibody is preferably detectably labeled and/orbound to a solid support.

[0024] In another embodiment, the present invention concerns animmune-related disease diagnostic kit, comprising an anti-PRO antibodyand a carrier in suitable packaging. The kit preferably containsinstructions for using the antibody to detect the presence of the PROpolypeptide. Preferably the carrier is pharmaceutically acceptable.

[0025] In another embodiment, the present invention concerns adiagnostic kit, containing an anti-PRO antibody in suitable packaging.The kit preferably contains instructions for using the antibody todetect the PRO polypeptide.

[0026] In another embodiment, the invention provides a method ofdiagnosing an immune-related disease in a mammal which comprisesdetecting the presence or absence or a PRO polypeptide in a test sampleof tissue cells obtained from said mammal, wherein the presence orabsence of the PRO polypeptide in said test sample is indicative of thepresence of an immune-related disease in said mammal.

[0027] In another embodiment, the present invention concerns a methodfor identifying an agonist of a PRO polypeptide comprising:

[0028] (a) contacting cells and a test compound to be screened underconditions suitable for the induction of a cellular response normallyinduced by a PRO polypeptide; and

[0029] (b) determining the induction of said cellular response todetermine if the test compound is an effective agonist, wherein theinduction of said cellular response is indicative of said test compoundbeing an effective agonist.

[0030] In another embodiment, the invention concerns a method foridentifying a compound capable of inhibiting the activity of a PROpolypeptide comprising contacting a candidate compound with a PROpolypeptide under conditions and for a time sufficient to allow thesetwo components to interact and determining whether the activity of thePRO polypeptide is inhibited. In a specific aspect, either the candidatecompound or the PRO polypeptide is immobilized on a solid support. Inanother aspect, the non-immobilized component carries a detectablelabel. In a preferred aspect, this method comprises the steps of:

[0031] (a) contacting cells and a test compound to be screened in thepresence of a PRO polypeptide under conditions suitable for theinduction of a cellular response normally induced by a PRO polypeptide;and

[0032] (b) determining the induction of said cellular response todetermine if the test compound is an effective antagonist.

[0033] In another embodiment, the invention provides a method foridentifying a compound that inhibits the expression of a PRO polypeptidein cells that normally express the polypeptide, wherein the methodcomprises contacting the cells with a test compound and determiningwhether the expression of the PRO polypeptide is inhibited. In apreferred aspect, this method comprises the steps of:

[0034] (a) contacting cells and a test compound to be screened underconditions suitable for allowing expression of the PRO polypeptide; and

[0035] (b) determining the inhibition of expression of said polypeptide.

[0036] In yet another embodiment, the present invention concerns amethod for treating an immune-related disorder in a mammal that sufferstherefrom comprising administering to the mammal a nucleic acid moleculethat codes for either (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide or (c) an antagonist of a PRO polypeptide, wherein saidagonist or antagonist may be an anti-PRO antibody. In a preferredembodiment, the mammal is human. In another preferred embodiment, thenucleic acid is administered via ex vivo gene therapy. In a furtherpreferred embodiment, the nucleic acid is comprised within a vector,more preferably an adenoviral, adeno-associated viral, lentiviral orretroviral vector.

[0037] In yet another aspect, the invention provides a recombinant viralparticle comprising a viral vector consisting essentially of a promoter,nucleic acid encoding (a) a PRO polypeptide, (b) an agonist polypeptideof a PRO polypeptide, or (c) an antagonist polypeptide of a PROpolypeptide, and a signal sequence for cellular secretion of thepolypeptide, wherein the viral vector is in association with viralstructural proteins. Preferably, the signal sequence is from a mammal,such as from a native PRO polypeptide.

[0038] In a still further embodiment, the invention concerns an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) a PROpolypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) anantagonist polypeptide of a PRO polypeptide, and a signal sequence forcellular secretion of the polypeptide, wherein said producer cellpackages the retroviral vector in association with the structuralproteins to produce recombinant retroviral particles.

[0039] In a still further embodiment, the invention provides a methodfor increasing the infiltration of inflammatory cells from thevasculature into a tissue of a mammal comprising administering to saidmammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or(c) an antagonist of a PRO polypeptide, wherein the infiltration ofinflammatory cells from the vasculature in the mammal is increased.

[0040] In a still further embodiment, the invention provides a methodfor decreasing the infiltration of inflammatory cells from thevasculature into a tissue of a mammal comprising administering to saidmammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or(c) an antagonist of a PRO polypeptide, wherein the infiltration ofinflammatory cells from the vasculature in the mammal is decreased.

[0041] In a still further embodiment, the invention provides a method ofincreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theactivity of T-lymphocytes in the mammal is increased.

[0042] In a still further embodiment, the invention provides a method ofdecreasing the activity of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theactivity of T-lymphocytes in the mammal is decreased.

[0043] In a still further embodiment, the invention provides a method ofincreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theproliferation of T-lymphocytes in the mammal is increased.

[0044] In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes in a mammal comprisingadministering to said mammal (a) a PRO polypeptide, (b) an agonist of aPRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein theproliferation of T-lymphocytes in the mammal is decreased.

[0045] In a still further embodiment, the invention provides a method ofstimulating the proliferation of T-cells comprising contacting saidT-cells with a PRO1475, PRO5723, PRO7425, or PRO9940 polypeptide oragonist thereof, wherein said T-cell proliferation is stimulated.

[0046] In a still further embodiment, the invention provides a method ofdecreasing the proliferation of T-lymphocytes comprising contacting saidT-lymphocytes with a PRO1265, PRO1308 or PRO4405 polypeptide, whereinthe proliferation of T-lymphocytes is decreased.

[0047] B. Additional Embodiments

[0048] In other embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

[0049] In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

[0050] In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

[0051] In yet other embodiments, the invention provides oligonucleotideprobes useful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

[0052] In other embodiments, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes a PROpolypeptide.

[0053] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91 % nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule encoding a PRO polypeptide having afull-length amino acid sequence as disclosed herein, an amino acidsequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein, or(b) the complement of the DNA molecule of (a).

[0054] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO polypeptide cDNA as disclosed herein, the codingsequence of a PRO polypeptide lacking the signal peptide as disclosedherein, the coding sequence of an extracellular domain of atransmembrane PRO polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

[0055] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

[0056] Another aspect the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO polypeptides are contemplated.

[0057] Another embodiment is directed to fragments of a PRO polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PROpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments are usually at least about 20nucleotides in length, alternatively at least about 30 nucleotides inlength, alternatively at least about 40 nucleotides in length,alternatively at least about 50 nucleotides in length, alternatively atleast about 60 nucleotides in length, alternatively at least about 70nucleotides in length, alternatively at least about 80 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 100 nucleotides in length, alternatively atleast about 110 nucleotides in length, alternatively at least about 120nucleotides in length, alternatively at least about 130 nucleotides inlength, alternatively at least about 140 nucleotides in length,alternatively at least about 150 nucleotides in length, alternatively atleast about 160 nucleotides in length, alternatively at least about 170nucleotides in length, alternatively at least about 180 nucleotides inlength, alternatively at least about 190 nucleotides in length,alternatively at least about 200 nucleotides in length, alternatively atleast about 250 nucleotides in length, alternatively at least about 300nucleotides in length, alternatively at least about 350 nucleotides inlength, alternatively at least about 400 nucleotides in length,alternatively at least about 450 nucleotides in length, alternatively atleast about 500 nucleotides in length, alternatively at least about 600nucleotides in length, alternatively at least about 700 nucleotides inlength, alternatively at least about 800 nucleotides in length,alternatively at least about 900 nucleotides in length and alternativelyat least about 1000 nucleotides in length, wherein in this context theterm “about” means the referenced nucleotide sequence length plus orminus 10% of that referenced length. It is noted that novel fragments ofa PRO polypeptide-encoding nucleotide sequence may be determined in aroutine manner by aligning the PRO polypeptide-encoding nucleotidesequence with other known nucleotide sequences using any of a number ofwell known sequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

[0058] In another embodiment, the invention provides isolated PROpolypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.

[0059] In a certain aspect, the invention concerns an isolated PROpolypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

[0060] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

[0061] In a specific aspect, the invention provides an isolated PROpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as herein before described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

[0062] Another aspect the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

[0063] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

[0064] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisecontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

[0065] In a still further embodiment, the invention concerns acomposition of matter comprising a PRO polypeptide, or an agonist orantagonist of a PRO polypeptide as herein described, or an anti-PROantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0066] Another embodiment of the present invention is directed to theuse of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe PRO polypeptide, an agonist or antagonist thereof or an anti-PROantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067]FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a nativesequence PRO 1265 cDNA, wherein SEQ ID NO:1 is a clone designated hereinas “DNA60764-1533”.

[0068]FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived fromthe coding sequence of SEQ ID NO:1 shown in FIG. 1.

[0069]FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a nativesequence PRO1308 cDNA, wherein SEQ ID NO:3 is a clone designated hereinas “DNA62306-1570”.

[0070]FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived fromthe coding sequence of SEQ ID NO:3 shown in FIG. 3.

[0071]FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a nativesequence PRO1475 cDNA, wherein SEQ ID NO:5 is a clone designated hereinas “DNA61185-1646”. FIG. 6 shows the amino acid sequence (SEQ ID NO:6)derived from the coding sequence of SEQ ID NO:5 shown in FIG. 5.

[0072]FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a nativesequence PRO4405 cDNA, wherein SEQ ID NO:7 is a clone designated hereinas “DNA84920-2614”. FIG. 8 shows the amino acid sequence (SEQ ID NO:8)derived from the coding sequence of SEQ ID NO:7 shown in FIG. 7.

[0073]FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a nativesequence PRO5723 cDNA, wherein SEQ ID NO:9 is a clone designated hereinas “DNA82361”. FIG. 10 shows the amino acid sequence (SEQ ID NO:10)derived from the coding sequence of SEQ ID NO:9 shown in FIG. 9.

[0074]FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a nativesequence PRO7425 cDNA, wherein SEQ ID NO:11 is a clone designated hereinas “DNA108792-2753”. FIG. 12 shows the amino acid sequence (SEQ IDNO:12) derived from the coding sequence of SEQ ID NO:11 shown in FIG.11.

[0075]FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a nativesequence PRO9940 cDNA, wherein SEQ ID NO:13 is a clone designated hereinas “DNA92282”. FIG. 14 shows the amino acid sequence (SEQ ID NO:14)derived from the coding sequence of SEQ ID NO:13 shown in FIG. 13.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] I. Definitions

[0077] The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods. Theterm “PRO polypeptide” refers to each individual PRO/number polypeptidedisclosed herein. All disclosures in this specification which refer tothe “PRO polypeptide” refer to each of the polypeptides individually aswell as jointly. For example, descriptions of the preparation of,purification of, derivation of, formation of antibodies to or against,administration of, compositions containing, treatment of a disease with,etc., pertain to each polypeptide of the invention individually. Theterm “PRO polypeptide” also includes variants of the PRO/numberpolypeptides disclosed herein.

[0078] A “native sequence PRO polypeptide” comprises a polypeptidehaving the same amino acid sequence as the corresponding PRO polypeptidederived from nature. Such native sequence PRO polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

[0079] The PRO polypeptide “extracellular domain” or “ECD” refers to aform of the PRO polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PROpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are contemplated by thepresent invention.

[0080] The approximate location of the “signal peptides” of the variousPRO polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

[0081] “PRO polypeptide variant” means an active PRO polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO polypeptide sequence asdisclosed herein, a PRO polypeptide sequence lacking the signal peptideas disclosed herein, an extracellular domain of a PRO polypeptide, withor without the signal peptide, as disclosed herein or any other fragmentof a full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20 aminoacids in length, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

[0082] “Percent (%) amino acid sequence identity” with respect to thePRO polypeptide sequences identified herein is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table I below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.OD. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0083] In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

[0084] 100 times the fraction X/Y

[0085] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations using this method, Tables 2 and 3 demonstrate howto calculate the % amino acid sequence identity of the amino acidsequence designated “Comparison Protein” to the amino acid sequencedesignated “PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

[0086] Unless specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan =1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

[0087] Percent amino acid sequence identity may also be determined usingthe sequence comparison program NCBI-BLAST2 (Altschul et al., NucleicAcids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparisonprogram may be downloaded from http://www.ncbi.nlm.nih.gov or otherwiseobtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0088] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

[0089] 100 times the fraction X/Y

[0090] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0091] “PRO variant polynucleotide” or “PRO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PROpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Ordinarily, a PRO variant polynucleotide will have atleast about 80% nucleic acid sequence identity, alternatively at leastabout 81% nucleic acid sequence identity, alternatively at least about82% nucleic acid sequence identity, alternatively at least about 83%nucleic acid sequence identity, alternatively at least about 84% nucleicacid sequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence PRO polypeptide sequence as disclosed herein, afull-length native sequence PRO polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

[0092] Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

[0093] “Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table I below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.OD. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0094] In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

[0095] 100 times the fraction W/Z

[0096] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

[0097] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

[0098] Percent nucleic acid sequence identity may also be determinedusing the sequence comparison program NCBI-BLAST2 (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequencecomparison program may be downloaded from http://www.ncbi.nlm.nih.gov orotherwise obtained from the National Institute of Health, Bethesda, MD.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0099] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

[0100] 100 times the fraction W/Z

[0101] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0102] In other embodiments, PRO variant polynucleotides are nucleicacid molecules that encode an active PRO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

[0103] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0104] An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

[0105] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0106] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0107] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-PRO monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies), anti-PROantibody compositions with polyepitopic specificity, single chainanti-PRO antibodies, and fragments of anti-PRO antibodies (see below).The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

[0108] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0109] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.00 15 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0110] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0111] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

[0112] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

[0113] “Active” or “activity” for the purposes herein refers to form(s)of a PRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

[0114] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

[0115] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

[0116] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

[0117] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0118] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0119] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0120] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0121] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0122] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and—binding site. This region consists of a dimer ofone heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0123] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab′ fragments by the addition of a few residuesat the carboxy terminus of the heavy chain CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0124] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains.

[0125] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0126] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0127] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0128] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0129] An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

[0130] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

[0131] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0132] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

[0133] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons.

[0134] The term “immune related disease” means a disease in which acomponent of the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

[0135] The term “T cell mediated disease” means a disease in which Tcells directly or indirectly mediate or otherwise contribute to amorbidity in a mammal. The T cell mediated disease may be associatedwith cell mediated effects, lymphokine mediated effects, etc., and eveneffects associated with B cells if the B cells are stimulated, forexample, by the lymphokines secreted by T cells.

[0136] Examples of immune-related and inflammatory diseases, some ofwhich are immune or T cell mediated, which can be treated according tothe invention include systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenia purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft -versus-host-disease. Infectious diseases includingviral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, andE, herpes, etc., bacterial infections, fungal infections, protozoalinfections and parasitic infections.

[0137] The term “effective amount” is a concentration or amount of a PROpolypeptide and/or agonist/antagonist which results in achieving aparticular stated purpose. An “effective amount” of a PRO polypeptide oragonist or antagonist thereof may be determined empirically.Furthermore, a “therapeutically effective amount” is a concentration oramount of a PRO polypeptide and/or agonist/antagonist which is effectivefor achieving a stated therapeutic effect. This amount may also bedetermined empirically.

[0138] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof.

[0139] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

[0140] A “growth inhibitory agent” when used herein refers to a compoundor composition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce GI arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13.

[0141] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

[0142] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM. TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is averageof EQ  * B is average of ND  * match with stop is _M; stop-stop = 0; J(joker) match = 0  */ #define _M −8 /* value of a match with a stop */int _day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W XY Z */ /* A */ {2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1, −2, −1, 0, _M, 1,0, −2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4, 3, 2, −5, 0, 1, −2,0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C */ {−2,−4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5, −4, 0,−2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2, 0, 0,−4, −3, 2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4, 2}, /* E */ {0, 2, −5,3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0,−4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0, −4, _M,−5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ {1, 0, −3, 1, 0, −5, 5,−2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0, −5, 0}, /*H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, _M, 0, 3, 2, −1,−1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3, −2, 5, 0,−2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J */ {0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1, _M, −1, 1,3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3, 2, −4, −2,2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0, 2, −2, 0, −1, −2} /* M*/ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2, −1, 0, −2,−1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2, −2, 0, 1,−3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py;   /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy= 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis + = (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =−ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “(%d %s%s)”, ngapx, (dna)?“base”: “residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “% s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy == 1)?“”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score: %d(match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2++) { nn = stripname(namex[i]); if (nn >lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } *put out a number line: dumpblock()  */ static nums(ix) nums int  ix; /*index in out[] holding seq line */ { char nline[P_LINE]; register i, j;register char *pn, *px, *py; for(pn = nline, i = 0; i < lmax+P_SPC; i++,pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) { if(*py == ‘ ’ || *py == ‘−’); *pn = ‘ ’; else { if (i%10 == 0 || (i == 1&& nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j; j/= 10, px−−)*px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’; i++; } } *pn =‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx);(void) putc(‘\n’, fx); } /*  * put out a line (name, [num], seq. [num]):dumpblock()  */ static putline(ix) putline int   ix; { ...putline int i;register char *px; for (px = namex[ix], i = 0; *px && *px != ‘:’; px++,i++) (void) putc(*px, fx); for (;i < lmax+P_SPC; i++) (void) putc(‘ ’,fx); /* these count from 1:  * ni[] is current element (from 1)  * nc[]is number at start of current line  */ for (px = out[ix]; *px; px++)(void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /*  * put a line ofstars (seqs always in out[0], out[1]): dumpblock()  */ static stars()stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0]== ‘ ’ && *(p0[0]) == ‘ ’) || !*out[1] || (*out[1] == ‘ ’ && *(po[1]) ==‘ ’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0− ‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align()  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup() --cleanup any tmp file  * getseq() -- read in seq, set dna, len, maxlen  *g_calloc() -- calloc() with error checkin  * readjmps() -- get the goodjmps, from tmp file if necessary  * writejmps() -- write a filled arrayof jmps to a tmp file: nw()  */ #include “nw.h” #include <sys/file.h>char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */ FILE *fj; intcleanup(); /* cleanup tmp file */ long lseek(); /*  * remove any tmpfile if we blow  */ cleanup(i) cleanup int i; { if (fj) (void)unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna, len,maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upper orlower case  */ char * getseq(file, len) getseq char *file; /* file name*/ int *len; /* seq len */ { char line[1024], *pseq; register char *px,*py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc() failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’;*py = ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc() failed %s (n= %d, sz= %d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[] or tmp file, set pp[], reset dmax: main()  */ readjmps() readjmps {int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open() %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1= 0, dmax0 = dmax, xx = len0; ;i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmps if(j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0);(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[il] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[il] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; il++; } else if (siz > 0) { /* gapin first seq */ pp[0] .n[i0] = siz; pp[0] .x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

[0143] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonProtein XXXXXYYYYYYY (Length = 12 amino acids) % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

[0144] TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonProtein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

[0145] TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides) % nucleic acidsequence identity = (the number of identically matching nucleotidesbetween the two nucleic acid sequences as determined by ALIGN-2) dividedby (the total number of nucleotides of the PRO-DNA nucleic acidsequence) = 6 divided by 14 = 42.9%

[0146] TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence) = 4 dividedby 12 = 33.3%

[0147] II. Compositions and Methods of the Invention

[0148] A. Full-Length PRO Polypeptides

[0149] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO polypeptides. In particular, cDNAs encoding variousPRO polypeptides have been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologous andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

[0150] As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

[0151] B. PRO Polypeptide Variants

[0152] In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

[0153] Variations in the native full-length sequence PRO or in variousdomains of the PRO described herein, can be made, for example, using anyof the techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO.Optionally, the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the PRO.Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the PRO with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

[0154] PRO polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

[0155] PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

[0156] In particular embodiments, conservative substitutions of interestare shown in Table 6 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucineleu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln;asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leuPro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucineleu

[0157] Substantial modifications in function or immunological identityof the PRO polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0158] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0159] (2) neutral hydrophilic: cys, ser, thr;

[0160] (3) acidic: asp, glu;

[0161] (4) basic: asn, gin, his, lys, arg;

[0162] (5) residues that influence chain orientation: gly, pro; and

[0163] (6) aromatic: trp, tyr, phe.

[0164] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0165] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

[0166] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., New York); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0167] C. Modifications of PRO

[0168] Covalent modifications of PRO are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C- terminal residues of the PRO. Derivatization withbifunctional agents is useful, for instance, for crosslinking PRO to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0169] Other modifications include deamidation of glutarianyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0170] Another type of covalent modification of the PRO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

[0171] Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0172] Another means of increasing the number of carbohydrate moietieson the PRO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0173] Removal of carbohydrate moieties present on the PRO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0174] Another type of covalent modification of PRO comprises linkingthe PRO polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0175] The PRO of the present invention may also be modified in a way toform a chimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

[0176] In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl- terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an alpha-tubulin epitope peptide[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad.Sci. USA, 87:6393-6397 (1990)].

[0177] In an alternative embodiment, the chimeric molecule may comprisea fusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0178] D. Preparation of PRO

[0179] The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

[0180] 1. Isolation of DNA Encoding PRO

[0181] DNA encoding PRO may be obtained from a cDNA library preparedfrom tissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

[0182] Libraries can be screened with probes (such as antibodies to thePRO or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0183] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0184] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined using methods known in the art and as described herein.

[0185] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0186] 2. Selection and Transformation of Host Cells

[0187] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

[0188] Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearraicroinjection, electroporation, bacterial protoplast fusion withintact cells, or polycations, e.g., polybrene, polyornithine, may alsobe used. For various techniques for transforming mammalian cells, seeKeown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour etal., Nature, 336:348-352 (1988).

[0189] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. hicheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3 110 may be modified toeffect a genetic mutation in the genes encoding proteins endogenous tothe host, with examples of such hosts including E. coli W3110 strain1A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4,which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7(ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac) 169 degP ompT kan^(r) ; E. coli W3 110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

[0190] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forPRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host mnicroorganism. Others include Schizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published May 2,1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0191] Suitable host cells for the expression of glycosylated PRO arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

[0192] 3. Selection and Use of a Replicable Vector

[0193] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

[0194] The PRO may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveronyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmanmmalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

[0195] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0196] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0197] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0198] Expression and cloning vectors usually contain a promoteroperably linked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S. D.) sequence operably linked to the DNA encoding PRO.

[0199] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0200] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0201] PRO transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, and fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0202] Transcription of a DNA encoding the PRO by higher eukaryotes maybe increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

[0203] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO.

[0204] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO in E recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0205] 4. Detecting Gene Amplification/Expression

[0206] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0207] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

[0208] 5. Purification of Polypeptide

[0209] Forms of PRO may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

[0210] It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

[0211] E. Tissue Distribution

[0212] The location of tissues expressing the PRO can be identified bydetermining mRNA expression in various human tissues. The location ofsuch genes provides information about which tissues are most likely tobe affected by the stimulating and inhibiting activities of the PROpolypeptides. The location of a gene in a specific tissue also providessample tissue for the activity blocking assays discussed below.

[0213] As noted before, gene expression in various tissues may bemeasured by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

[0214] Gene expression in various tissues, alternatively, may bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids may beeither monoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceof a PRO polypeptide or against a synthetic peptide based on the DNAsequences encoding the PRO polypeptide or against an exogenous sequencefused to a DNA encoding a PRO polypeptide and encoding a specificantibody epitope. General techniques for generating antibodies, andspecial protocols for Northern blotting and in situ hybridization areprovided below.

[0215] F. Antibody Binding Studies

[0216] The activity of the PRO polypeptides can be further verified byantibody binding studies, in which the ability of anti-PRO antibodies toinhibit the effect of the PRO polypeptides, respectively, on tissuecells is tested. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies, the preparationof which will be described hereinbelow.

[0217] Antibody binding studies may be carried out in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

[0218] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of target protein in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies preferably are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

[0219] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0220] For immunohistochemistry, the tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

[0221] G. Cell-Based Assays

[0222] Cell-based assays and animal models for immune related diseasescan be used to further understand the relationship between the genes andpolypeptides identified herein and the development and pathogenesis ofimmune related disease.

[0223] In a different approach, cells of a cell type known to beinvolved in a particular immune related disease are transfected with thecDNAs described herein, and the ability of these cDNAs to stimulate orinhibit immune function is analyzed. Suitable cells can be transfectedwith the desired gene, and monitored for immune function activity. Suchtransfected cell lines can then be used to test the ability of poly- ormonoclonal antibodies or antibody compositions to inhibit or stimulateimmune function, for example to modulate T-cell proliferation orinflammatory cell infiltration. Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of immune related diseases.

[0224] In addition, primary cultures derived from transgenic animals (asdescribed below) can be used in the cell-based assays herein, althoughstable cell lines are preferred. Techniques to derive continuous celllines from transgenic animals are well known in the art (see, e.g.,Small et al., Mol. Cell. Biol. 5: 642-648 [1985]).

[0225] One suitable cell based assay is the mixed lymphocyte reaction(MLR). Current Protocols in Immunology, unit 3.12; edited by J EColigan, A M Kruisbeek, D H Marglies, E M Shevach, W Strober, NationalInstitutes of Health, Published by John Wiley & Sons, Inc. In thisassay, the ability of a test compound to stimulate or inhibit theproliferation of activated T cells is assayed. A suspension of responderT cells is cultured with allogeneic stimulator cells and theproliferation of T cells is measured by uptake of tritiated thymidine.This assay is a general measure of T cell reactivity. Since the majorityof T cells respond to and produce IL-2 upon activation, differences inresponsiveness in this assay in part reflect differences in IL-2production by the responding cells. The MLR results can be verified by astandard lymphokine (IL-2) detection assay. Current Protocols inImmunology, above, 3.15, 6.3.

[0226] A proliferative T cell response in an MLR assay may be due todirect mitogenic properties of an assayed molecule or to externalantigen induced activation. Additional verification of the T cellstimulatory activity of the PRO polypeptides can be obtained by acostimulation assay. T cell activation requires an antigen specificsignal mediated through the T-cell receptor (TCR) and a costimulatorysignal mediated through a second ligand binding interaction, forexample, the B7 (CD80, CD86)/CD28 binding interaction. CD28 crosslinkingincreases lymphokine secretion by activated T cells. T cell activationhas both negative and positive controls through the binding of ligandswhich have a negative or positive effect. CD28 and CTLA-4 are relatedglycoproteins in the Ig superfamily which bind to B7. CD28 binding to B7has a positive costimulation effect of T cell activation; conversely,CTLA-4 binding to B7 has a negative T cell deactivating effect.Chambers, C. A. and Allison, J. P., Curr. Opin. Immunol. (1997) 9:396.Schwartz, R. H., Cell (1992) 71:1065; Linsey, P. S. and Ledbetter, J.A., Annu. Rev. Immunol. (1993) 11:191; June, C. H. et al, Immunol. Today(1994) 15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulationassay, the PRO polypeptides are assayed for T cell costimulatory orinhibitory activity.

[0227] PRO polypeptides, as well as other compounds of the invention,which are stimulators (costimulators) of T cell proliferation andagonists, e.g., agonist antibodies, thereto as determined by MLR andcostimulation assays, for example, are useful in treating immune relateddiseases characterized by poor, suboptimal or inadequate immunefunction. These diseases are treated by stimulating the proliferationand activation of T cells (and T cell mediated immunity) and enhancingthe immune response in a mammal through administration of a stimulatorycompound, such as the stimulating PRO polypeptides. The stimulatingpolypeptide may, for example, be a PRO polypeptide or an agonistantibody thereof.

[0228] Direct use of a stimulating compound as in the invention has beenvalidated in experiments with 4-1BB glycoprotein, a member of the tumornecrosis factor receptor family, which binds to a ligand (4-1BBL)expressed on primed T cells and signals T cell activation and growth.Alderson, M. E. et al., J. Immunol. (1994) 24:2219.

[0229] The use of an agonist stimulating compound has also beenvalidated experimentally. Activation of 4-1BB by treatment with anagonist anti-4-1 BB antibody enhances eradication of tumors. Hellstrom,I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998) 18:1. Immunoadjuvanttherapy for treatment of tumors, described in more detail below, isanother example of the use of the stimulating compounds of theinvention.

[0230] An immune stimulating or enhancing effect can also be achieved byantagonizing or blocking the activity of a PRO which has been found tobe inhibiting in the MLR assay. Negating the inhibitory activity of thecompound produces a net stimulatory effect. Suitableantagonists/blocking compounds are antibodies or fragments thereof whichrecognize and bind to the inhibitory protein, thereby blocking theeffective interaction of the protein with its receptor and inhibitingsignaling through the receptor. This effect has been validated inexperiments using anti-CTLA-4 antibodies which enhance T cellproliferation, presumably by removal of the inhibitory signal caused byCTLA-4 binding. Walunas, T. L. et al, Immunity (1994) 1:405.

[0231] Alternatively, an immune stimulating or enhancing effect can alsobe achieved by administration of a PRO which has vascular permeabilityenhancing properties. Enhanced vacuolar permeability would be beneficialto disorders which can be attenuated by local infiltration of immunecells (e.g., monocytes, eosinophils, PMNs) and inflammation.

[0232] On the other hand, PRO polypeptides, as well as other compoundsof the invention, which are direct inhibitors of T cellproliferation/activation, lymphokine secretion, and/or vascularpermeability can be directly used to suppress the immune response. Thesecompounds are useful to reduce the degree of the immune response and totreat immune related diseases characterized by a hyperactive,superoptimal, or autoimmune response. This use of the compounds of theinvention has been validated by the experiments described above in whichCTLA-4 binding to receptor B7 deactivates T cells. The direct inhibitorycompounds of the invention function in an analogous manner. The use ofcompound which suppress vascular permeability would be expected toreduce inflammation. Such uses would be beneficial in treatingconditions associated with excessive inflammation.

[0233] Alternatively, compounds, e.g., antibodies, which bind tostimulating PRO polypeptides and block the stimulating effect of thesemolecules produce a net inhibitory effect and can be used to suppressthe T cell mediated immune response by inhibiting T cellproliferation/activation and/or lymphokine secretion. Blocking thestimulating effect of the polypeptides suppresses the immune response ofthe mammal. This use has been validated in experiments using an anti-IL2antibody. In these experiments, the antibody binds to IL2 and blocksbinding of IL2 to its receptor thereby achieving a T cell inhibitoryeffect.

[0234] H. Animal Models

[0235] The results of the cell based in vitro assays can be furtherverified using in vivo animal models and assays for T-cell function. Avariety of well known animal models can be used to further understandthe role of the genes identified herein in the development andpathogenesis of immune related disease, and to test the efficacy ofcandidate therapeutic agents, including antibodies, and otherantagonists of the native polypeptides, including small moleculeantagonists. The in vivo nature of such models makes them predictive ofresponses in human patients. Animal models of immune related diseasesinclude both non-recombinant and recombinant (transgenic) animals.Non-recombinant animal models include, for example, rodent, e.g., murinemodels. Such models can be generated by introducing cells into syngeneicmice using standard techniques, e.g., subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, etc.

[0236] Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. Graft-versus-host disease models provide a means of assessing Tcell reactivity against MHC antigens and minor transplant antigens. Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.3.

[0237] An animal model for skin allograft rejection is a means oftesting the ability of T cells to mediate in vivo tissue destruction anda measure of their role in transplant rejection. The most common andaccepted models use murine tail-skin grafts. Repeated experiments haveshown that skin allograft rejection is mediated by T cells, helper Tcells and killer-effector T cells, and not antibodies. Auchincloss, H.Jr. and Sachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed.,Raven Press, New York, 1989, 889-992. A suitable procedure is describedin detail in Current Protocols in Immunology, above, unit 4.4. Othertransplant rejection models which can be used to test the compounds ofthe invention are the allogeneic heart transplant models described byTanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al,J. Immunol. (1994) 4330-4338.

[0238] Animal models for delayed type hypersensitivity provides an assayof cell mediated immune function as well. Delayed type hypersensitivityreactions are a T cell mediated in vivo immune response characterized byinflammation which does not reach a peak until after a period of timehas elapsed after challenge with an antigen. These reactions also occurin tissue specific autoimmune diseases such as multiple sclerosis (MS)and experimental autoimmune encephalomyelitis (EAE, a model for MS). Asuitable procedure is described in detail in Current Protocols inImmunology, above, unit 4.5.

[0239] EAE is a T cell mediated autoimmune disease characterized by Tcell and mononuclear cell inflammation and subsequent demyelination ofaxons in the central nervous system. EAE is generally considered to be arelevant animal model for MS in humans. Bolton, C., Multiple Sclerosis(1995) 1:143. Both acute and relapsing-remitting models have beendeveloped. The compounds of the invention can be tested for T cellstimulatory or inhibitory activity against immune mediated demyelinatingdisease using the protocol described in Current Protocols in Immunology,above, units 15.1 and 15.2. See also the models for myelin disease inwhich oligodendrocytes or Schwann cells are grafted into the centralnervous system as described in Duncan, I. D. et al, Molec. Med. Today(1997) 554-561.

[0240] Contact hypersensitivity is a simple delayed typehypersensitivity in vivo assay of cell mediated immune function. In thisprocedure, cutaneous exposure to exogenous haptens which gives rise to adelayed type hypersensitivity reaction which is measured andquantitated. Contact sensitivity involves an initial sensitizing phasefollowed by an elicitation phase. The elicitation phase occurs when theT lymphocytes encounter an antigen to which they have had previouscontact. Swelling and inflammation occur, making this an excellent modelof human allergic contact dermatitis. A suitable procedure is describedin detail in Current Protocols in Immunology, Eds. J. E. Cologan, A. M.Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley &Sons, Inc., 1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun.Today 19 (1): 37-44 (1998) .

[0241] An animal model for arthritis is collagen-induced arthritis. Thismodel shares clinical, histological and immunological characteristics ofhuman autoimmune rheumatoid arthritis and is an acceptable model forhuman autoimmune arthritis. Mouse and rat models are characterized bysynovitis, erosion of cartilage and subchondral bone. The compounds ofthe invention can be tested for activity against autoimmune arthritisusing the protocols described in Current Protocols in Immunology, above,units 15.5. See also the model using a monoclonal antibody to CD18 andVLA-4 integrins described in Issekutz, A. C. et al., Immunology (1996)88:569.

[0242] A model of asthma has been described in which antigen-inducedairway hyper-reactivity, pulmonary eosinophilia and inflammation areinduced by sensitizing an animal with ovalbumin and then challenging theanimal with the same protein delivered by aerosol. Several animal models(guinea pig, rat, non-human primate) show symptoms similar to atopicasthma in humans upon challenge with aerosol antigens. Murine modelshave many of the features of human asthma. Suitable procedures to testthe compounds of the invention for activity and effectiveness in thetreatment of asthma are described by Wolyniec, W. W. et al, Am. J.Respir. Cell Mol. Biol. (1998) 18:777 and the references cited therein.

[0243] Additionally, the compounds of the invention can be tested onanimal models for psoriasis like diseases. Evidence suggests a T cellpathogenesis for psoriasis. The compounds of the invention can be testedin the scid/scid mouse model described by Schon, M. P. et al, Nat. Med.(1997) 3:183, in which the mice demonstrate histopathologic skin lesionsresembling psoriasis. Another suitable model is the human skin/scidmouse chimera prepared as described by Nickoloff, B. J. et al, Am. J.Path. (1995) 146:580.

[0244] Recombinant (transgenic) animal models can be engineered byintroducing the coding portion of the genes identified herein into thegenome of animals of interest, using standard techniques for producingtransgenic animals. Animals that can serve as a target for transgenicmanipulation include, without limitation, mice, rats, rabbits, guineapigs, sheep, goats, pigs, and non-human primates, e.g., baboons,chimpanzees and monkeys. Techniques known in the art to introduce atransgene into such animals include pronucleic microinjection (Hoppe andWanger, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82,6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal., Cell 56, 313-321 [1989]); electroporation of embryos (Lo, Mol. Cel.Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano etal., Cell 57, 717-73 [1989]). For review, see, for example, U.S. Pat.No. 4,736,866.

[0245] For the purpose of the present invention, transgenic animalsinclude those that carry the transgene only in part of their cells(“mosaic animals”). The transgene can be integrated either as a singletransgene, or in concatamers, e.g., head-to-head or head-to-tailtandems. Selective introduction of a transgene into a particular celltype is also possible by following, for example, the technique of Laskoet al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).

[0246] The expression of the transgene in transgenic animals can bemonitored by standard techniques. For example, Southern blot analysis orPCR amplification can be used to verify the integration of thetransgene. The level of mRNA expression can then be analyzed usingtechniques such as in situ hybridization, Northern blot analysis, PCR,or immunocytochemistry.

[0247] The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated withthe compounds of the invention to determine the extent of the T cellproliferation stimulation or inhibition of the compounds. In theseexperiments, blocking antibodies which bind to the PRO polypeptide,prepared as described above, are administered to the animal and theeffect on immune function is determined.

[0248] Alternatively, “knock out” animals can be constructed which havea defective or altered gene encoding a polypeptide identified herein, asa result of homologous recombination between the endogenous geneencoding the polypeptide and altered genomic DNA encoding the samepolypeptide introduced into an embryonic cell of the animal. Forexample, cDNA encoding a particular polypeptide can be used to clonegenomic DNA encoding that polypeptide in accordance with establishedtechniques. A portion of the genomic DNA encoding a particularpolypeptide can be deleted or replaced with another gene, such as a geneencoding a selectable marker which can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi,Cell, 51:503 (1987) for a description of homologous recombinationvectors]. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected [see e.g.,Li et al., Cell, 69:915 (1992)]. The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

[0249] I. ImmunoAdjuvant Therapy

[0250] In one embodiment, the immunostimulating compounds of theinvention can be used in immunoadjuvant therapy for the treatment oftumors (cancer). It is now well established that T cells recognize humantumor specific antigens. One group of tumor antigens, encoded by theMAGE, BAGE and GAGE families of genes, are silent in all adult normaltissues, but are expressed in significant amounts in tumors, such asmelanomas, lung tumors, head and neck tumors, and bladder carcinomas.DeSmet, C. et al., (1996) Proc. Natl. Acad. Sci. USA, 93:7149. It hasbeen shown that costimulation of T cells induces tumor regression and anantitumor response both in vitro and in vivo. Melero, I. et al., NatureMedicine (1997) 3:682; Kwon, E. D. et al., Proc. Natl. Acad. Sci. USA(1997) 94: 8099; Lynch, D. H. et al, Nature Medicine (1997) 3:625; Finn,0. J. and Lotze, M. T., J. Immunol. (1998) 21:114. The stimulatorycompounds of the invention can be administered as adjuvants, alone ortogether with a growth regulating agent, cytotoxic agent orchemotherapeutic agent, to stimulate T cell proliferation/activation andan antitumor response to tumor antigens. The growth regulating,cytotoxic, or chemotherapeutic agent may be administered in conventionalamounts using known administration regimes. Immunostimulating activityby the compounds of the invention allows reduced amounts of the growthregulating, cytotoxic, or chemotherapeutic agents thereby potentiallylowering the toxicity to the patient.

[0251] J. Screening Assays for Drug Candidates

[0252] Screening assays for drug candidates are designed to identifycompounds that bind to or complex with the polypeptides encoded by thegenes identified herein or a biologically active fragment thereof, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds, including peptides, preferably soluble peptides,(poly)peptide-immunoglobulin fusions, and, in particular, antibodiesincluding, without limitation, poly- and monoclonal antibodies andantibody fragments, single-chain antibodies, anti-idiotypic antibodies,and chimeric or humanized versions of such antibodies or fragments, aswell as human antibodies and antibody fragments. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art. All assays are commonin that they call for contacting the drug candidate with a polypeptideencoded by a nucleic acid identified herein under conditions and for atime sufficient to allow these two components to interact.

[0253] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the polypeptide and drying. Alternatively, an immobilizedantibody, e.g., a monoclonal antibody, specific for the polypeptide tobe immobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labelledantibody specifically binding the immobilized complex.

[0254] If the candidate compound interacts with but does not bind to aparticular protein encoded by a gene identified herein, its interactionwith that protein can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers [Fieldsand Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA 88, 9578-9582 (1991)] as disclosed by Chevray andNathans, Proc. NatL. Acad. Sci. USA 89, 5789-5793 (1991). Manytranscriptional activators, such as yeast GALA, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GALA-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

[0255] In order to find compounds that interfere with the interaction ofa gene identified herein and other intra- or extracellular componentscan be tested, a reaction mixture is usually prepared containing theproduct of the gene and the intra- or extracellular component underconditions and for a time allowing for the interaction and binding ofthe two products. To test the ability of a test compound to inhibitbinding, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. The binding (complex formation)between the test compound and the intra- or extracellular componentpresent in the mixture is monitored as described above. The formation ofa complex in the control reaction(s) but not in the reaction mixturecontaining the test compound indicates that the test compound interfereswith the interaction of the test compound and its reaction partner.

[0256] K. Compositions and Methods for the Treatment of Immune RelatedDiseases

[0257] The compositions useful in the treatment of immune relateddiseases include, without limitation, proteins, antibodies, smallorganic molecules, peptides, phosphopeptides, antisense and ribozymemolecules, triple helix molecules, etc. that inhibit or stimulate immunefunction, for example, T cell proliferation/activation, lymphokinerelease, or immune cell infiltration.

[0258] For example, antisense RNA and RNA molecules act to directlyblock the translation of mRNA by hybridizing to targeted mRNA andpreventing protein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0259] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g., Rossi, Current Biology 4, 469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0260] Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g.,PCT publication No. WO 97/33551, supra.

[0261] These molecules can be identified by any or any combination ofthe screening assays discussed above and/or by any other screeningtechniques well known for those skilled in the art.

[0262] L. Anti-PRO Antibodies

[0263] The present invention further provides anti-PRO antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

[0264] 1. Polyclonal Antibodies

[0265] The anti-PRO antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0266] 2. Monoclonal Antibodies

[0267] The anti-PRO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0268] The immunizing agent will typically include the PRO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0269] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0270] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst PRO. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0271] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0272] The monoclonal antibodies secreted by the subdlones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0273] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0274] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0275] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0276] 3. Human and Humanized Antibodies

[0277] The anti-PRO antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0278] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0279] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boemer et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0280] The antibodies may also be affinity matured using known selectionand/or mutagenesis methods as described above. Preferred affinitymatured antibodies have an affinity which is five times, more preferably10 times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

[0281] 4. Bispecific Antibodies

[0282] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0283] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0284] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0285] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0286] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)2 fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0287] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0288] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodieswith more than two valencies are contemplated. For example, trispecificantibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0289] Exemplary bispecific antibodies may bind to two differentepitopes on a given PRO polypeptide herein. Alternatively, an anti-PROpolypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular PRO polypeptide.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular PRO polypeptide. These antibodiespossess a PRO-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPFA, DOTA, or TETA. Anotherbispecific antibody of interest binds the PRO polypeptide and furtherbinds tissue factor (TF).

[0290] 5. Heteroconjugate Antibodies

[0291] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0292] 6. Effector Function Engineering

[0293] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0294] 7. Immunoconjugates

[0295] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0296] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

[0297] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipiridate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See W094/11026.

[0298] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0299] 8. Immunoliposomes

[0300] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0301] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0302] M. Pharmaceutical Compositions

[0303] The active PRO molecules of the invention (e.g., PROpolypeptides, anti-PRO antibodies, and/or variants of each) as well asother molecules identified by the screening assays disclosed above, canbe administered for the treatment of immune related diseases, in theform of pharmaceutical compositions.

[0304] Therapeutic formulations of the active PRO molecule, preferably apolypeptide or antibody of the invention, are prepared for storage bymixing the active molecule having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0305] Compounds identified by the screening assays disclosed herein canbe formulated in an analogous manner, using standard techniques wellknown in the art.

[0306] Lipofections or liposomes can also be used to deliver the PROmolecule into cells. Where antibody fragments are used, the smallestinhibitory fragment which specifically binds to the binding domain ofthe target protein is preferred. For example, based upon the variableregion sequences of an antibody, peptide molecules can be designed whichretain the ability to bind the target protein sequence. Such peptidescan be synthesized chemically and/or produced by recombinant DNAtechnology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90,7889-7893 [1993]).

[0307] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

[0308] The active PRO molecules may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0309] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0310] Sustained-release preparations or the PRO molecules may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S-S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0311] N. Methods of Treatment

[0312] It is contemplated that the polypeptides, antibodies and otheractive compounds of the present invention may be used to treat variousimmune related diseases and conditions, such as T cell mediateddiseases, including those characterized by infiltration of inflammatorycells into a tissue, stimulation of T-cell proliferation, inhibition ofT-cell proliferation, increased or decreased vascular permeability orthe inhibition thereof.

[0313] Exemplary conditions or disorders to be treated with thepolypeptides, antibodies and other compounds of the invention, include,but are not limited to systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, osteoarthritis,spondyloarthropathies, systemic sclerosis (scleroderma), idiopathicinflammatory myopathies (dermatomyositis, polymyositis), Sjögren'ssyndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenia purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease.

[0314] In systemic lupus erythematosus, the central mediator of diseaseis the production of auto-reactive antibodies to self proteins/tissuesand the subsequent generation of immune-mediated inflammation.Antibodies either directly or indirectly mediate tissue injury. Though Tlymphocytes have not been shown to be directly involved in tissuedamage, T lymphocytes are required for the development of auto-reactiveantibodies. The genesis of the disease is thus T lymphocyte dependent.Multiple organs and systems are affected clinically including kidney,lung, musculoskeletal system, mucocutaneous, eye, central nervoussystem, cardiovascular system, gastrointestinal tract, bone marrow andblood.

[0315] Rheumatoid arthritis (RA) is a chronic systemic autoimmuneinflammatory disease that mainly involves the synovial membrane ofmultiple joints with resultant injury to the articular cartilage. Thepathogenesis is T lymphocyte dependent and is associated with theproduction of rheumatoid factors, auto-antibodies directed against selfIgG, with the resultant formation of immune complexes that attain highlevels in joint fluid and blood. These complexes in the joint may inducethe marked infiltrate of lymphocytes and monocytes into the synovium andsubsequent marked synovial changes; the joint space/fluid if infiltratedby similar cells with the addition of numerous neutrophils. Tissuesaffected are primarily the joints, often in symmetrical pattern.However, extra-articular disease also occurs in two major forms. Oneform is the development of extra-articular lesions with ongoingprogressive joint disease and typical lesions of pulmonary fibrosis,vasculitis, and cutaneous ulcers. The second form of extra-articulardisease is the so called Felty's syndrome which occurs late in the RAdisease course, sometimes after joint disease has become quiescent, andinvolves the presence of neutropenia, thrombocytopenia and splenomegaly.This can be accompanied by vasculitis in multiple organs with formationsof infarcts, skin ulcers and gangrene. Patients often also developrheumatoid nodules in the subcutis tissue overlying affected joints; thenodules late stage have necrotic centers surrounded by a mixedinflammatory cell infiltrate. Other manifestations which can occur in RAinclude: pericarditis, pleuritis, coronary arteritis, intestitialpneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, andrhematoid nodules.

[0316] Juvenile chronic arthritis is a chronic idiopathic inflammatorydisease which begins often at less than 16 years of age. Its phenotypehas some similarities to RA; some patients which are rhematoid factorpositive are classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and istypically destructive and leads to joint ankylosis and retarded growth.Other manifestations can include chronic anterior uveitis and systemicamyloidosis.

[0317] Spondyloarthropathies are a group of disorders with some commonclinical features and the common association with the expression ofHLA-B27 gene product. The disorders include: ankylosing sponylitis,Reiter's syndrome (reactive arthritis), arthritis associated withinflammatory bowel disease, spondylitis associated with psoriasis,juvenile onset spondyloarthropathy and undifferentiatedspondyloarthropathy. Distinguishing features include sacroileitis withor without spondylitis; inflammatory asymmetric arthritis; associationwith HLA-B27 (a serologically defined allele of the HLA-B locus of classI MHC); ocular inflammation, and absence of autoantibodies associatedwith other rheumatoid disease. The cell most implicated as key toinduction of the disease is the CD8 +T lymphocyte, a cell which targetsantigen presented by class I MHC molecules. CD8+T cells may reactagainst the class I MHC allele HLA-B27 as if it were a foreign peptideexpressed by MHC class I molecules. It has been hypothesized that anepitope of HLA-B27 may mimic a bacterial or other microbial antigenicepitope and thus induce a CD8+T cells response.

[0318] Systemic sclerosis (scleroderma) has an unknown etiology. Ahallmark of the disease is induration of the skin; likely this isinduced by an active inflammatory process. Scleroderma can be localizedor systemic; vascular lesions are common and endothelial cell injury inthe microvasculature is an early and important event in the developmentof systemic sclerosis; the vascular injury may be immune mediated. Animmunologic basis is implied by the presence of mononuclear cellinfiltrates in the cutaneous lesions and the presence of anti-nuclearantibodies in many patients. ICalif.M-1 is often upregulated on the cellsurface of fibroblasts in skin lesions suggesting that T cellinteraction with these cells may have a role in the pathogenesis of thedisease. Other organs involved include: the gastrointestinal tract:smooth muscle atrophy and fibrosis resulting in abnormalperistalsis/motility; kidney: concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries withresultant reduced renal cortical blood flow, results in proteinuria,azotemia and hypertension; skeletal muscle: atrophy, interstitialfibrosis; inflammation; lung: interstitial pneumonitis and interstitialfibrosis; and heart: contraction band necrosis, scarring/fibrosis.

[0319] Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components, proteinsand RNA's, involved in protein synthesis.

[0320] Sjögren's syndrome is due to immune-mediated inflammation andsubsequent functional destruction of the tear glands and salivaryglands. The disease can be associated with or accompanied byinflammatory connective tissue diseases. The disease is associated withautoantibody production against Ro and La antigens, both of which aresmall RNA-protein complexes. Lesions result in keratoconjunctivitissicca, xerostomia, with other manifestations or associations includingbilary cirrhosis, peripheral or sensory neuropathy, and palpablepurpura.

[0321] Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc., particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

[0322] Sarcoidosis is a condition of unknown etiology which ischaracterized by the presence of epithelioid granulomas in nearly anytissue in the body; involvement of the lung is most common. Thepathogenesis involves the persistence of activated macrophages andlymphoid cells at sites of the disease with subsequent chronic sequelaeresultant from the release of locally and systemically active productsreleased by these cell types.

[0323] Autoimmune hemolytic anemia including autoimmune hemolyticanemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria is aresult of production of antibodies that react with antigens expressed onthe surface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

[0324] In autoimmune thrombocytopenia including thrombocytopeniapurpura, and immune-mediated thrombocytopenia in other clinicalsettings, platelet destruction/removal occurs as a result of eitherantibody or complement attaching to platelets and subsequent removal bycomplement lysis, ADCC or FC-receptor mediated mechanisms.

[0325] Thyroiditis including Grave's disease, Hashimoto's thyroiditis,juvenile lymphocytic thyroiditis, and atrophic thyroiditis, are theresult of an autoimmune response against thyroid antigens withproduction of antibodies that react with proteins present in and oftenspecific for the thyroid gland. Experimental models exist includingspontaneous models: rats (BUF and BB rats) and chickens (obese chickenstrain); inducible models: immunization of animals with eitherthyroglobulin, thyroid microsomal antigen (thyroid peroxidase).

[0326] Type I diabetes mellitus or insulin-dependent diabetes is theautoimmune destruction of pancreatic islet β cells; this destruction ismediated by auto-antibodies and auto-reactive T cells. Antibodies toinsulin or the insulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

[0327] Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

[0328] Demyelinating diseases of the central and peripheral nervoussystems, including Multiple Sclerosis; idiopathic demyelinatingpolyneuropathy or Guillain-Barre syndrome; and Chronic InflammatoryDemyelinating Polyneuropathy, are believed to have an autoimmune basisand result in nerve demyelination as a result of damage caused tooligodendrocytes or to myelin directly. In MS there is evidence tosuggest that disease induction and progression is dependent on Tlymphocytes. Multiple Sclerosis is a demyelinating disease that is Tlymphocyte-dependent and has either a relapsing-remitting course or achronic progressive course. The etiology is unknown; however, viralinfections, genetic predisposition, environment, and autoimmunity allcontribute. Lesions contain infiltrates of predominantly T lymphocytemediated, microglial cells and infiltrating macrophages; CD4+Tlymphocytes are the predominant cell type at lesions. The mechanism ofoligodendrocyte cell death and subsequent demyelination is not known butis likely T lymphocyte driven.

[0329] Inflammatory and Fibrotic Lung Disease, including EosinophilicPneumonias; Idiopathic Pulmonary Fibrosis, and HypersensitivityPneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit.

[0330] Autoimmune or Immune-mediated Skin Disease including Bullous SkinDiseases, Erythema Multiforme, and Contact Dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

[0331] Psoriasis is a T lymphocyte-mediated inflammatory disease.Lesions contain infiltrates of T lymphocytes, macrophages and antigenprocessing cells, and some neutrophils.

[0332] Allergic diseases, including asthma; allergic rhinitis; atopicdermatitis; food hypersensitivity; and urticaria are T lymphocytedependent. These diseases are predominantly mediated by T lymphocyteinduced inflammation, IgE mediated-inflammation or a combination ofboth.

[0333] Transplantation associated diseases, including Graft rejectionand Graft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent;inhibition of T lymphocyte function is ameliorative. Other diseases inwhich intervention of the immune and/or inflammatory response havebenefit are infectious disease including but not limited to viralinfection (including but not limited to AIDS, hepatitis A, B, C, D, Eand herpes) bacterial infection, fungal infections, and protozoal andparasitic infections (molecules (or derivatives/agonists) whichstimulate the MLR can be utilized therapeutically to enhance the immuneresponse to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e., as from chemotherapy) immunodeficiency, and neoplasia.

[0334] It has been demonstrated that some human cancer patients developan antibody and/or T lymphocyte response to antigens on neoplasticcells. It has also been shown in animal models of neoplasia thatenhancement of the immune response can result in rejection or regressionof that particular neoplasm. Molecules that enhance the T lymphocyteresponse in the MLR have utility in vivo in enhancing the immuneresponse against neoplasia. Molecules which enhance the T lymphocyteproliferative response in the MLR (or small molecule agonists orantibodies that affected the same receptor in an agonistic fashion) canbe used therapeutically to treat cancer. Molecules that inhibit thelymphocyte response in the MLR also function in vivo during neoplasia tosuppress the immune response to a neoplasm; such molecules can either beexpressed by the neoplastic cells themselves or their expression can beinduced by the neoplasm in other cells. Antagonism of such inhibitorymolecules (either with antibody, small molecule antagonists or othermeans) enhances immune-mediated tumor rejection.

[0335] Additionally, inhibition of molecules with proinflammatoryproperties may have therapeutic benefit in reperfusion injury; stroke;myocardial infarction; atherosclerosis; acute lung injury; hemorrhagicshock; burn; sepsis/septic shock; acute tubular necrosis; endometriosis;degenerative joint disease and pancreatis.

[0336] The compounds of the present invention, e.g., polypeptides orantibodies, are administered to a mammal, preferably a human, in accordwith known methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation (intranasal,intrapulmonary) routes. Intravenous or inhaled administration ofpolypeptides and antibodies is preferred.

[0337] In immunoadjuvant therapy, other therapeutic regimens, suchadministration of an anti-cancer agent, may be combined with theadministration of the proteins, antibodies or compounds of the instantinvention. For example, the patient to be treated with a theimmunoadjuvant of the invention may also receive an anti-cancer agent(chemotherapeutic agent) or radiation therapy. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the immunoadjuvant or may be givensimultaneously therewith. Additionally, an anti-oestrogen compound suchas tamoxifen or an anti-progesterone such as onapristone (see, EP616812) may be given in dosages known for such molecules.

[0338] It may be desirable to also administer antibodies against otherimmune disease associated or tumor associated antigens, such asantibodies which bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4,or vascular endothelial factor (VEGF). Alternatively, or in addition,two or more antibodies binding the same or two or more differentantigens disclosed herein may be coadministered to the patient.Sometimes, it may be beneficial to also administer one or more cytokinesto the patient. In one embodiment, the PRO polypeptides arecoadministered with a growth inhibitory agent. For example, the growthinhibitory agent may be administered first, followed by a PROpolypeptide. However, simultaneous administration or administrationfirst is also contemplated. Suitable dosages for the growth inhibitoryagent are those presently used and may be lowered due to the combinedaction (synergy) of the growth inhibitory agent and the PRO polypeptide.

[0339] For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments.

[0340] For example, depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of polypeptide orantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. A typical daily dosage might range from about1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

[0341] O. Articles of Manufacture

[0342] In another embodiment of the invention, an article of manufacturecontaining materials (e.g., comprising a PRO molecule) useful for thediagnosis or treatment of the disorders described above is provided. Thearticle of manufacture comprises a container and an instruction.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for diagnosing or treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide or an antibody of the invention. An instruction or label on,or associated with, the container indicates that the composition is usedfor diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

[0343] P. Diagnosis and Prognosis of Immune Related Disease

[0344] Cell surface proteins, such as proteins which are overexpressedin certain immune related diseases, are excellent targets for drugcandidates or disease treatment. The same proteins along with secretedproteins encoded by the genes amplified in immune related disease statesfind additional use in the diagnosis and prognosis of these diseases.For example, antibodies directed against the protein products of genesamplified in multiple sclerosis, rheumatoid arthritis, or another immunerelated disease, can be used as diagnostics or prognostics.

[0345] For example, antibodies, including antibody fragments, can beused to qualitatively or quantitatively detect the expression ofproteins encoded by amplified or overexpressed genes (“marker geneproducts”). The antibody preferably is equipped with a detectable, e.g.,fluorescent label, and binding can be monitored by light microscopy,flow cytometry, fluorimetry, or other techniques known in the art. Thesetechniques are particularly suitable, if the overexpressed gene encodesa cell surface protein Such binding assays are performed essentially asdescribed above.

[0346] In situ detection of antibody binding to the marker gene productscan be performed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

[0347] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0348] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0349] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, VA.

EXAMPLE 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

[0350] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

[0351] Using this extracellular domain homology screen, consensus DNAsequences were assembled relative to the other identified EST sequencesusing phrap. In addition, the consensus DNA sequences obtained wereoften (but not always) extended using repeated cycles of BLAST orBLAST-2 and phrap to extend the consensus sequence as far as possibleusing the sources of EST sequences discussed above.

[0352] Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0353] The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif.. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

EXAMPLE 2 Isolation of cDNA Clones Using Specific Database Query

[0354] Polypeptide-encoding nucleic acid sequences were identified byusing the computer program BLAST or BLAST2 (Altschul et al., Methods inEnzymology 266:460-480 (1996)) to search for homologues of specificgenes in public (e.g., GenBank) and/or private (LIFESEQ®, IncytePharmaceuticals, Inc., Palo Alto, Calif.) databases In this instance,investigators used a single previously identified gene as a querysequence to search for related homologues. The significance of thehomology was determined on a case by case basis for each gene. When asignificant homology was found, this resulted in the identification ofadditional EST sequences which either corresponded to full-lengthclones, which were examined and sequenced or served as a template forthe creation of cloning oligonucleotides which were then used to screenvarious tissue libraries resulting in isolation of DNA encoding a nativesequence PRO polypeptide.

EXAMPLE 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

[0355] Various polypeptide-encoding nucleic acid sequences wereidentified by applying a proprietary signal sequence finding algorithmdeveloped by Genentech, Inc. (South San Francisco, Calif.) upon ESTs aswell as clustered and assembled EST fragments from public (e.g.,GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., PaloAlto, Calif.) databases. The signal sequence algorithm computes asecretion signal score based on the character of the DNA nucleotidessurrounding the first and optionally the second methionine codon(s)(ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

EXAMPLE 4 Isolation of cDNA Clones Encoding Human PRO Polypeptides

[0356] Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO polypeptides asdisclosed herein. These cDNAs were then deposited under the terms of theBudapest Treaty with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA (ATCC) as shown in Table7 below. TABLE 7 Material UNQ PRO ATCC # ATCC Deposit Date DNA60764-1533636 1265 203452 November 10, 1998 DNA62306-1570 674 1308 203254September 9, 1998 DNA61185-1646 746 1475 203464 November 17, 1998DNA84920-2614 1930  4405 203966 April 27, 1999 DNA82361 1972  5723 N/D*DNA108792-2753 2966  7425 PTA-617 August 31, 1999 DNA92282 889 9940 N/D*

EXAMPLE 5 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay(No. 24)

[0357] This example shows that the polypeptides of the invention areactive as stimulators of the proliferation of T-lymphocytes. Compoundswhich stimulate proliferation of lymphocytes are useful therapeuticallywhere enhancement of an immune response is beneficial. A therapeuticagent may also take the form of antagonists of the PRO polypeptides ofthe invention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide, which would be expected to inhibitT-lymphocyte proliferation.

[0358] The basic protocol for this assay is described in CurrentProtocols in Inumunology, unit 3.12; edited by J. E. Coligan, A. M.Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, NationalInstitutes of Health, Published by John Wiley & Sons, Inc.

[0359] More specifically, in one assay variant, peripheral bloodmononuclear cells (PBMC) are isolated from mammalian individuals, forexample a human volunteer, by leukopheresis (one donor will supplystimulator PBMCs, the other donor will supply responder PBMCs). Ifdesired, the cells are frozen in fetal bovine serum and DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37° C.,5% CO₂) and then washed and resuspended to 3×10⁶ cells/ml of assay media(RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine,1% HEPES, 1% non-essential amino acids, 1% pyruvate).

[0360] The stimulator PBMCs are prepared by irradiating the cells (about3000 Rads). The assay is prepared by plating in triplicate wells amixture of: 100 μl of test sample diluted to 1% or to 0.1%; 50 μl ofirradiated stimulator cells and 50 μl of responder PBMC cells. 100microliters of cell culture media or 100 microliter of CD4-IgG is usedas the control. The wells are then incubated at 37° C., 5% CO₂ for 4days. On day 5 and each well is pulsed with tritiated thymidine (1.0mCi/well; Amersham). After 6 hours the cells are washed 3 times and thenthe uptake of the label is evaluated.

[0361] In another variant of this assay, PBMCs are isolated from thespleens of Balb/c mice and C57B6 mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above. The results of this assayfor compounds of the invention are shown below in Table 8. Positiveincreases over control are considered positive with increases of greaterthan or equal to 180% being preferred. However, any value greater thancontrol indicates a stimulatory effect for the test protein. TABLE 8 PROPRO Concentration Percent Increase Over Control PRO1475  0.07 nM 137.5PRO1475  0.70 nM 243.0 PRO5723 66.00 nM 187.8 PRO7425  6.30 nM 313.8PRO9940 80.15 nM 183.3 PRO9940 80.15 nM 244.6

EXAMPLE 6 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay(No. 67)

[0362] This example shows that one or more of the PRO polypeptides areactive as inhibitors of the proliferation of stimulated T-lymphocytes.Compounds which inhibit proliferation of lymphocytes are usefultherapeutically where suppression of an immune response is beneficial.

[0363] The basic protocol for this assay is described in CurrentProtocols in Immunology, unit 3.12; edited by J. E. Coligan, A. M.Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, NationalInstitutes of Health, Published by John Wiley & Sons, Inc.

[0364] More specifically, in one assay variant, peripheral bloodmononuclear cells (PBMC) are isolated from mammalian individuals, forexample a human volunteer, by leukopheresis (one donor will supplystimulator PBMCs, the other donor will supply responder PBMCs). Ifdesired, the cells are frozen in fetal bovine serum and DMSO afterisolation. Frozen cells may be thawed overnight in assay media (37° C.,5% CO₂) and then washed and resuspended to 3×10⁶ cells/ml of assay media(RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine,1% HEPES, 1% non-essential amino acids, 1% pyruvate). The stimulatorPBMCs are prepared by irradiating the cells (about 3000 Rads).

[0365] The assay is prepared by plating in triplicate wells a mixtureof:

[0366] 100:1 of test sample diluted to 1% or to 0.1%,

[0367] 50:1 of irradiated stimulator cells, and

[0368] 50:1 of responder PBMC cells.

[0369] 100 microliters of cell culture media or 100 microliter ofCD4-IgG is used as the control. The wells are then incubated at 37° C.,5% CO₂ for 4 days. On day 5, each well is pulsed with tritiatedthymidine (1.0 mCi/well; Amersham). After 6 hours the cells are washed 3times and then the uptake of the label is evaluated.

[0370] In another variant of this assay, PBMCs are isolated from thespleens of Balb/c mice and C57B6 mice. The cells are teased from freshlyharvested spleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

[0371] Any decreases below control is considered to be a positive resultfor an inhibitory compound, with decreases of less than or equal to 80%being preferred. However, any value less than control indicates aninhibitory effect for the test protein. Results are given in Table 9.TABLE 9 PRO PRO Concentration Percent Decrease Below Control PRO1265 3.2nM 79.3 PRO1265  32 nM 69.6 PRO1308 1.1 nM 70.5 PRO1308  11 nM 64.4PRO4405 0.63 nM  80.1 PRO4405 6.3 nM 22.7

EXAMPLE 7 In situ Hybridization

[0372] In situ hybridization is a powerful and versatile technique forthe detection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

[0373] In situ hybridization was performed following an optimizedversion of the protocol by Lu and Gillett, Cell Vision 1: 169-176(1994), using PCR-generated ³³P-labeled riboprobes. Briefly,formalin-fixed, paraffin-embedded human tissues were sectioned,deparaffinized, deproteinated in proteinase K (20 mg/ml) for 15 minutesat 37° C., and further processed for in situ hybridization as describedby Lu and Gillett, supra. A [³³p] UTP-labeled antisense riboprobe wasgenerated from a PCR product and hybridized at 55° C. overnight. Theslides were dipped in Kodak NTB2 nuclear track emulsion and exposed for4 weeks.

[0374]³³P-Riboprobe Synthesis

[0375] 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol)were speed vac dried. To each tube containing dried³³P-UTP, thefollowing ingredients were added: 2.0 μl 5× transcription buffer; 1.0 μlDTT (100 mM); 2.0 μl NTP mix (2.5 mM: 10 μl; each of 10 mM GTP, CTP &ATP+10 μl H ₂O); 1.0 μl UTP (50 μM); 1.0 μl Rnasin; 1.0 μl DNA template(1 μg); 1.0 μl H₂O.

[0376] The tubes were incubated at 37° C. for one hour. 1.0 μRQ1 DNasewere added, followed by incubation at 37° C. for 15 minutes. 90 μl TE(10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture waspipetted onto DE81 paper. The remaining solution was loaded in aMicrocon-50 ultrafiltration unit, and spun using program 10 (6 minutes).The filtration unit was inverted over a second tube and spun usingprogram 2 (3 minutes). After the final recovery spin, 100 III TE wereadded. 1 μl of the final product was pipetted on DE81 paper and countedin 6 ml of Biofluor II.

[0377] The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 IIIof RNA Mrk III were added to 3 μl of loading buffer. After heating on a95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, the sample loaded, and run at180-250 volts for 45 minutes. The gel was wrapped in saran wrap andexposed to XAR film with an intensifying screen in −70° C. freezer onehour to overnight.

[0378]³³P-Hybridization

[0379] Pretreatment of frozen sections The slides were removed from thefreezer, placed on aluminum trays and thawed at room temperature for 5minutes. The trays were placed in 55° C. incubator for five minutes toreduce condensation. The slides were fixed for 10 minutes in 4%paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5minutes, at room temperature (25 ml 20×SSC+975 ml SQ H₂O). Afterdeproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), thesections were washed in 0.5×SSC for 10 minutes at room temperature. Thesections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.

[0380] Pretreatment of paraffin-embedded sections The slides weredeparaffinized, placed in SQ H₂O, and rinsed twice in 2×SSC at roomtemperature, for 5 minutes each time. The sections were deproteinated in20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNasebuffer; 37° C., 15 minutes )—human embryo, or 8×proteinase K (100 μl in250 ml Rnase buffer, 37° C., 30 minutes)—formalin tissues. Subsequentrinsing in 0.5×SSC and dehydration were performed as described above.

[0381] Prehybridization The slides were laid out in plastic box linedwith Box buffer (4×SSC, 50% formamide)—saturated filter paper. Thetissue was covered with 50 μl of hybridization buffer (3.75 g DextranSulfate+6 ml SQ H₂O), vortexed and heated in the microwave for 2 minuteswith the cap loosened. After cooling on ice, 18.75 ml formamide, 3.75 ml20×SSC and 9 ml SQ H₂O were added, the tissue was vortexed well, andincubated at 42° C. for 1-4 hours.

[0382] Hybridization 1.0×10⁶ cp. probe and 1.0 μl RNA (50 mg/ml stock)per slide were heated at 95° C. for 3 minutes. The slides were cooled onice, and 48 μl hybridization buffer were added per slide. Aftervortexing, 50 μl ³³p mix were added to 50 μl prehybridization on slide.The slides were incubated overnight at 55° C.

[0383] Washes Washing was done 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25M EDTA, V_(f=4)L), followed byRNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlRnase buffer—20 μg/ml), The slides were washed 2×10 minutes with 2×SSC,EDTA at room temperature. The stringency wash conditions were asfollows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA,V_(f)=4L).

[0384] Alternatively, multi-tissue blots containing poly A⁺RNA (2 μg perlane) from various human tissues were purchased from Clontech (PaloAlto, Calif.). DNA probes were labeled with [α-³²P]dCTP by randompriming DNA labeling Beads (Pharmacia Biotech). Hybridization wasperformed with Expresshyb (Clontech) at 68° C. for 1 hr. The blots werethen washed with 2×SSC/0.05% SDS solution at room temperature for 40min, followed by washes in 0.1X SSC/0.1% SDS solution at 55° C. for 40min with one change of fresh solution. The blots were exposed in aphosphorimager.

[0385] This method was used to determine gene expression, analyze thetissue distribution of transcription, and follow changes in specificmRNA synthesis for the genes/DNAs and the proteins of the invention indiseased tissues isolated from human individuals suffering from aspecific disease. These results show more specifically where in diseasedtissues the genes of the invention are expressed and are more predictiveof the particular localization of the therapeutic effect of theinhibitory or stimulatory compounds of the invention (and agonists orantagonists thereof) in a disease. Hybridization is performed accordingto the method above described using one or more of the following tissueand cell samples:

[0386] (a) lymphocytes and antigen presenting cells (dendritic cells,Langherhans cells, macrophages and monocytes, NK cells);

[0387] (b) lymphoid tissues: normal and reactive lymph node, thymus,Bronchial Associated Lymphoid Tissues, (BALT), Mucosal AssociatedLymphoid Tissues (MALT);

[0388] (c) human disease tissues:

[0389] Synovium and joint of patients with Arthritis and DegenerativeJoint Disease;

[0390] Colon from patients with Inflammatory Bowel Disease includingUlcerative Colitis and Crohns disease;

[0391] Skin lesions from Psoriasis and other forms of dermatitis;

[0392] Lung tissue including BALT and tissue lymph nodes from chronicand acute bronchitis, pneumonia, pneumonitis, pleuritis;

[0393] Lung tissue including BALT and tissue lymph nodes from Asthma;

[0394] nasal and sinus tissue from patients with rhinitis or sinusitis;

[0395] Brain and Spinal cord from Multiple Sclerosis. Alzheimer'sDisease and Stroke;

[0396] Kidney from Nephritis, Glomerulonephritis and Systemic LupusErythematosis;

[0397] Liver from Infectious and non-infectious Hepatitis andacetaminophen-induced liver cirrhosis;

[0398] Tissues from Neoplasms/Cancer.

[0399] Expression is observed in one or more cell or tissue samplesindicating localization of the therapeutic effect of the compounds ofthe invention (and agonists or antagonists thereof) in the diseaseassociated with the cell or tissue sample.

[0400] Expression of DNA60764-1533 in Tumors

[0401] Fifteen lung tumors were examined (8 adeno and 7 squamouscarcinomas). Most tumors show some expression of DNA60764-1533.Expression is largely confined to mononuclear cells adjacent to theinfiltrating tumor. In one specific tumor (26A3), a squamous carcinoma,there appears to be expression by malignant epithelium. In anotherinstance, there is expression of DNA60764-1533 over mononuclear cells indamaged renal interstitium and in interstitial cells in a renal cellcarcinoma. There is expression of DNA60764-1533 over cells in fetalthymic medulla. Expression is found over cells in a germinal center,consistent with the fact that most FIG. 1 positive cells are probablyinflammatory in origin. The following probes were used for the in situstudy. 5′-GGATTCTAATACGACTCACTATAGGGCCGCGCTGTCCTGCTGTCACCA-3′ (6O764.p1SEQ ID NO: 15) 5′-CTATGAAATTAACCCTCACTAAAGGGAGTTCCCCTCCCCGAGAAGATA-3′(6O764.p2 SEQ ID NO: 16)

EXAMPLE 8 Use of PRO as a Hybridization Probe

[0402] The following method describes use of a nucleotide sequenceencoding PRO as a hybridization probe.

[0403] DNA comprising the coding sequence of full-length or mature PROas disclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

[0404] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO-derived probe to the filters isperformed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

[0405] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

EXAMPLE 9 Expression of PRO in E. coli

[0406] This example illustrates preparation of an unglycosylated form ofPRO by recombinant expression in E. coli.

[0407] The DNA sequence encoding PRO is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO coding region, lambdatranscriptional terminator, and an argU gene.

[0408] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0409] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0410] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0411] PRO may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO is initially amplifiedusing selected PCR primers. The primers will contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3 110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate·2H2O, 1.07 g KCI, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0412]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 mil QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0413] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/ml. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros R1/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher acetonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0414] Fractions containing the desired folded PRO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

[0415] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

EXAMPLE 10 Expression of PRO in Mammalian Cells

[0416] This example illustrates preparation of a potentiallyglycosylated form of PRO by recombinant expression in mammalian cells.

[0417] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of thePRO DNA using ligation methods such as described in Sambrook et al.,supra. The resulting vector is called pRK5-PRO.

[0418] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaC2. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 mil of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0419] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/mi ³⁵S-cysteine and 200 μCi/mi 35S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

[0420] In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

[0421] In another embodiment, PRO can be expressed in CHO cells. ThepRK5-PRO can be transfected into CHO cells using known reagents such asCaPO4 or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO can then be concentrated and purified byany selected method.

[0422] Epitope-tagged PRO may also be expressed in host CHO cells. ThePRO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedPRO insert can then be subcloned into a SV40 promoter/enhancercontaining vector containing a selection marker such as DHFR forselection of stable clones. Finally, the CHO cells can be transfected(as described above) with the SV40 promoter/enhancer containing vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

[0423] PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

[0424] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgGl constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

[0425] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0426] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0427] The ampules containing the plasmid DNA are thawed by placementinto water bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 Jim diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, pH is determined. On day 1, thespinner is sampled and sparging with filtered air is commenced. On day2, the spinner is sampled, the temperature shifted to 33° C., and 30 mLof 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35%polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion)taken. Throughout the production, the pH is adjusted as necessary tokeep it at around 7.2. After 10 days, or until the viability droppedbelow 70%, the cell culture is harvested by centrifugation and filteringthrough a 0.22 μm filter. The filtrate was either stored at 4° C. orimmediately loaded onto columns for purification.

[0428] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0429] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 l of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0430] Many of me PRO polypeptides disclosed herein were successfullyexpressed as described above.

EXAMPLE 11 Expression of PRO in Yeast

[0431] The following method describes recombinant expression of PRO inyeast.

[0432] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

[0433] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0434] Recombinant PRO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

[0435] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

EXAMPLE 12 Expression of PRO in Baculovirus-Infected Insect Cells

[0436] The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

[0437] The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0438] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0439] Expressed poly-his tagged PRO can then be purified, for example,by Ni²⁺-chelate affinity chromatography as follows. Extracts areprepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.4 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A2w with loading buffer, at which pointfraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO are pooled and dialyzed againstloading buffer.

[0440] Alternatively, purification of the IgG tagged (or Fc tagged) PROcan be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

[0441] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

EXAMPLE 13 Preparation of Antibodies that Bind PRO

[0442] This example illustrates preparation of monoclonal antibodieswhich can specifically bind PRO.

[0443] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO, fusion proteins containingPRO, and cells expressing recombinant PRO on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

[0444] Mice, such as Balb/c, are immunized with the PRO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

[0445] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0446] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO is within the skill in theart.

[0447] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

EXAMPLE 14: Purification of PRO Polypeptides Using Specific Antibodies

[0448] Native or recombinant PRO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-PRO polypeptide, mature PRO polypeptide, or pre-PROpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling the anti-PROpolypeptide antibody to an activated chromatographic resin.

[0449] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0450] Such an immunoaffinity column is utilized in the purification ofPRO polypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0451] A soluble PRO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of PRO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

EXAMPLE 15 Drug Screening

[0452] This invention is particularly useful for screening compounds byusing PRO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The PRO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

[0453] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a PRO polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an PRO polypeptide or fragment thereof and assaying (I) for thepresence of a complex between the agent and the PRO polypeptide orfragment, or (ii) for the presence of a complex between the PROpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO polypeptide or fragment istypically labeled. After suitable incubation, free PRO polypeptide orfragment is separated from that present in bound form, and the amount offree or uncomplexed label is a measure of the ability of the particularagent to bind to PRO polypeptide or to interfere with the PROpolypeptide/cell complex.

[0454] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published onSeptember 13, 1984. Briefly stated, large numbers of different smallpeptide test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. As applied to a PRO polypeptide, thepeptide test compounds are reacted with PRO polypeptide and washed.Bound PRO polypeptide is detected by methods well known in the art.Purified PRO polypeptide can also be coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

[0455] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

EXAMPLE 16 Rational Drug Design

[0456] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a PRO polypeptide)or of small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 2: 19-21 (1991)).

[0457] In one approach, the three-dimensional structure of the PROpolypeptide, or of an PRO polypeptide-inhibitor complex, is determinedby x-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

[0458] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0459] By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

[0460] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 14 1 1844 DNA Homo Sapien 1 gacagtggag ggcagtggag aggaccgcgctgtcctgctg tcaccaagag 50 ctggagacac catctcccac cgagagtcat ggccccattggccctgcacc 100 tcctcgtcct cgtccccatc ctcctcagcc tggtggcctc ccaggactgg150 aaggctgaac gcagccaaga ccccttcgag aaatgcatgc aggatcctga 200ctatgagcag ctgctcaagg tggtgacctg ggggctcaat cggaccctga 250 agccccagagggtgattgtg gttggcgctg gtgtggccgg gctggtggcc 300 gccaaggtgc tcagcgatgctggacacaag gtcaccatcc tggaggcaga 350 taacaggatc gggggccgca tcttcacctaccgggaccag aacacgggct 400 ggattgggga gctgggagcc atgcgcatgc ccagctctcacaggatcctc 450 cacaagctct gccagggcct ggggctcaac ctgaccaagt tcacccagta500 cgacaagaac acgtggacgg aggtgcacga agtgaagctg cgcaactatg 550tggtggagaa ggtgcccgag aagctgggct acgccttgcg tccccaggaa 600 aagggccactcgcccgaaga catctaccag atggctctca accaggccct 650 caaagacctc aaggcactgggctgcagaaa ggcgatgaag aagtttgaaa 700 ggcacacgct cttggaatat cttctcggggaggggaacct gagccggccg 750 gccgtgcagc ttctgggaga cgtgatgtcc gaggatggcttcttctatct 800 cagcttcgcc gaggccctcc gggcccacag ctgcctcagc gacagactcc850 agtacagccg catcgtgggt ggctgggacc tgctgccgcg cgcgctgctg 900agctcgctgt ccgggcttgt gctgttgaac gcgcccgtgg tggcgatgac 950 ccagggaccgcacgatgtgc acgtgcagat cgagacctct cccccggcgc 1000 ggaatctgaa ggtgctgaaggccgacgtgg tgctgctgac ggcgagcgga 1050 ccggcggtga agcgcatcac cttctcgccgccgctgcccc gccacatgca 1100 ggaggcgctg cggaggctgc actacgtgcc ggccaccaaggtgttcctaa 1150 gcttccgcag gcccttctgg cgcgaggagc acattgaagg cggccactca1200 aacaccgatc gcccgtcgcg catgattttc tacccgccgc cgcgcgaggg 1250cgcgctgctg ctggcctcgt acacgtggtc ggacgcggcg gcagcgttcg 1300 ccggcttgagccgggaagag gcgttgcgct tggcgctcga cgacgtggcg 1350 gcattgcacg ggcctgtcgtgcgccagctc tgggacggca ccggcgtcgt 1400 caagcgttgg gcggaggacc agcacagccagggtggcttt gtggtacagc 1450 cgccggcgct ctggcaaacc gaaaaggatg actggacggtcccttatggc 1500 cgcatctact ttgccggcga gcacaccgcc tacccgcacg gctgggtgga1550 gacggcggtc aagtcggcgc tgcgcgccgc catcaagatc aacagccgga 1600aggggcctgc atcggacacg gccagccccg aggggcacgc atctgacatg 1650 gaggggcaggggcatgtgca tggggtggcc agcagcccct cgcatgacct 1700 ggcaaaggaa gaaggcagccaccctccagt ccaaggccag ttatctctcc 1750 aaaacacgac ccacacgagg acctcgcattaaagtatttt cggaaaaaaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaa 1844 2 567 PRT Homo Sapien 2 Met Ala Pro Leu Ala Leu His Leu LeuVal Leu Val Pro Ile Leu 1 5 10 15 Leu Ser Leu Val Ala Ser Gln Asp TrpLys Ala Glu Arg Ser Gln 20 25 30 Asp Pro Phe Glu Lys Cys Met Gln Asp ProAsp Tyr Glu Gln Leu 35 40 45 Leu Lys Val Val Thr Trp Gly Leu Asn Arg ThrLeu Lys Pro Gln 50 55 60 Arg Val Ile Val Val Gly Ala Gly Val Ala Gly LeuVal Ala Ala 65 70 75 Lys Val Leu Ser Asp Ala Gly His Lys Val Thr Ile LeuGlu Ala 80 85 90 Asp Asn Arg Ile Gly Gly Arg Ile Phe Thr Tyr Arg Asp GlnAsn 95 100 105 Thr Gly Trp Ile Gly Glu Leu Gly Ala Met Arg Met Pro SerSer 110 115 120 His Arg Ile Leu His Lys Leu Cys Gln Gly Leu Gly Leu AsnLeu 125 130 135 Thr Lys Phe Thr Gln Tyr Asp Lys Asn Thr Trp Thr Glu ValHis 140 145 150 Glu Val Lys Leu Arg Asn Tyr Val Val Glu Lys Val Pro GluLys 155 160 165 Leu Gly Tyr Ala Leu Arg Pro Gln Glu Lys Gly His Ser ProGlu 170 175 180 Asp Ile Tyr Gln Met Ala Leu Asn Gln Ala Leu Lys Asp LeuLys 185 190 195 Ala Leu Gly Cys Arg Lys Ala Met Lys Lys Phe Glu Arg HisThr 200 205 210 Leu Leu Glu Tyr Leu Leu Gly Glu Gly Asn Leu Ser Arg ProAla 215 220 225 Val Gln Leu Leu Gly Asp Val Met Ser Glu Asp Gly Phe PheTyr 230 235 240 Leu Ser Phe Ala Glu Ala Leu Arg Ala His Ser Cys Leu SerAsp 245 250 255 Arg Leu Gln Tyr Ser Arg Ile Val Gly Gly Trp Asp Leu LeuPro 260 265 270 Arg Ala Leu Leu Ser Ser Leu Ser Gly Leu Val Leu Leu AsnAla 275 280 285 Pro Val Val Ala Met Thr Gln Gly Pro His Asp Val His ValGln 290 295 300 Ile Glu Thr Ser Pro Pro Ala Arg Asn Leu Lys Val Leu LysAla 305 310 315 Asp Val Val Leu Leu Thr Ala Ser Gly Pro Ala Val Lys ArgIle 320 325 330 Thr Phe Ser Pro Pro Leu Pro Arg His Met Gln Glu Ala LeuArg 335 340 345 Arg Leu His Tyr Val Pro Ala Thr Lys Val Phe Leu Ser PheArg 350 355 360 Arg Pro Phe Trp Arg Glu Glu His Ile Glu Gly Gly His SerAsn 365 370 375 Thr Asp Arg Pro Ser Arg Met Ile Phe Tyr Pro Pro Pro ArgGlu 380 385 390 Gly Ala Leu Leu Leu Ala Ser Tyr Thr Trp Ser Asp Ala AlaAla 395 400 405 Ala Phe Ala Gly Leu Ser Arg Glu Glu Ala Leu Arg Leu AlaLeu 410 415 420 Asp Asp Val Ala Ala Leu His Gly Pro Val Val Arg Gln LeuTrp 425 430 435 Asp Gly Thr Gly Val Val Lys Arg Trp Ala Glu Asp Gln HisSer 440 445 450 Gln Gly Gly Phe Val Val Gln Pro Pro Ala Leu Trp Gln ThrGlu 455 460 465 Lys Asp Asp Trp Thr Val Pro Tyr Gly Arg Ile Tyr Phe AlaGly 470 475 480 Glu His Thr Ala Tyr Pro His Gly Trp Val Glu Thr Ala ValLys 485 490 495 Ser Ala Leu Arg Ala Ala Ile Lys Ile Asn Ser Arg Lys GlyPro 500 505 510 Ala Ser Asp Thr Ala Ser Pro Glu Gly His Ala Ser Asp MetGlu 515 520 525 Gly Gln Gly His Val His Gly Val Ala Ser Ser Pro Ser HisAsp 530 535 540 Leu Ala Lys Glu Glu Gly Ser His Pro Pro Val Gln Gly GlnLeu 545 550 555 Ser Leu Gln Asn Thr Thr His Thr Arg Thr Ser His 560 5653 2498 DNA Homo Sapien 3 cgtctctgcg ttcgccatgc gtcccggggc gccagggccactctggcctc 50 tgccctgggg ggccctggct tgggccgtgg gcttcgtgag ctccatgggc 100tcggggaacc ccgcgcccgg tggtgtttgc tggctccagc agggccagga 150 ggccacctgcagcctggtgc tccagactga tgtcacccgg gccgagtgct 200 gtgcctccgg caacattgacaccgcctggt ccaacctcac ccacccgggg 250 aacaagatca acctcctcgg cttcttgggccttgtccact gccttccctg 300 caaagattcg tgcgacggcg tggagtgcgg cccgggcaaggcgtgccgca 350 tgctgggggg ccgcccgcgc tgcgagtgcg cgcccgactg ctcggggctc400 ccggcgcggc tgcaggtctg cggctcagac ggcgccacct accgcgacga 450gtgcgagctg cgcgccgcgc gctgccgcgg ccacccggac ctgagcgtca 500 tgtaccggggccgctgccgc aagtcctgtg agcacgtggt gtgcccgcgg 550 ccacagtcgt gcgtcgtggaccagacgggc agcgcccact gcgtggtgtg 600 tcgagcggcg ccctgccctg tgccctccagccccggccag gagctttgcg 650 gcaacaacaa cgtcacctac atctcctcgt gccacatgcgccaggccacc 700 tgcttcctgg gccgctccat cggcgtgcgc cacgcgggca gctgcgcagg750 cacccctgag gagccgccag gtggtgagtc tgcagaagag gaagagaact 800tcgtgtgagc ctgcaggaca ggcctgggcc tggtgcccga ggccccccat 850 catcccctgttatttattgc cacagcagag tctaatttat atgccacgga 900 cactccttag agcccggattcggaccactt ggggatccca gaacctccct 950 gacgatatcc tggaaggact gaggaagggaggcctggggg ccggctggtg 1000 ggtgggatag acctgcgttc cggacactga gcgcctgatttagggccctt 1050 ctctaggatg ccccagcccc taccctaaga cctattgccg gggaggattc1100 cacacttccg ctcctttggg gataaaccta ttaattattg ctactatcaa 1150gagggctggg cattctctgc tggtaattcc tgaagaggca tgactgcttt 1200 tctcagccccaagcctctag tctgggtgtg tacggagggt ctagcctggg 1250 tgtgtacgga gggtctagcctgggtgagta cggagggtct agcctgggtg 1300 agtacggagg gtctagcctg ggtgagtacggagggtctag cctgggtgtg 1350 tatggaggat ctagcctggg tgagtatgga gggtctagcctgggtgagta 1400 tggagggtct agcctgggtg tgtatggagg gtctagcctg ggtgagtatg1450 gagggtctag cctgggtgtg tatggagggt ctagcctggg tgagtatgga 1500gggtctagcc tgggtgtgta cggagggtct agtctgagtg cgtgtgggga 1550 cctcagaacactgtgacctt agcccagcaa gccaggccct tcatgaaggc 1600 caagaaggct gccaccattccctgccagcc caagaactcc agcttcccca 1650 ctgcctctgt gtgccccttt gcgtcctgtgaaggccattg agaaatgccc 1700 agtgtgcccc ctgggaaagg gcacggcctg tgctcctgacacgggctgtg 1750 cttggccaca gaaccaccca gcgtctcccc tgctgctgtc cacgtcagtt1800 catgaggcaa cgtcgcgtgg tctcagacgt ggagcagcca gcggcagctc 1850agagcagggc actgtgtccg gcggagccaa gtccactctg ggggagctct 1900 ggcggggaccacgggccact gctcacccac tggccccgag gggggtgtag 1950 acgccaagac tcacgcatgtgtgacatccg gagtcctgga gccgggtgtc 2000 ccagtggcac cactaggtgc ctgctgcctccacagtgggg ttcacaccca 2050 gggctccttg gtcccccaca acctgccccg gccaggcctgcagacccaga 2100 ctccagccag acctgcctca cccaccaatg cagccggggc tggcgacacc2150 agccaggtgc tggtcttggg ccagttctcc cacgacggct caccctcccc 2200tccatctgcg ttgatgctca gaatcgccta cctgtgcctg cgtgtaaacc 2250 acagcctcagaccagctatg gggagaggac aacacggagg atatccagct 2300 tccccggtct ggggtgaggaatgtggggag cttgggcatc ctcctccagc 2350 ctcctccagc ccccaggcag tgccttacctgtggtgccca gaaaagtgcc 2400 cctaggttgg tgggtctaca ggagcctcag ccaggcagcccaccccaccc 2450 tggggccctg cctcaccaag gaaataaaga ctcaagccat aaaaaaaa2498 4 263 PRT Homo Sapien 4 Met Arg Pro Gly Ala Pro Gly Pro Leu Trp ProLeu Pro Trp Gly 1 5 10 15 Ala Leu Ala Trp Ala Val Gly Phe Val Ser SerMet Gly Ser Gly 20 25 30 Asn Pro Ala Pro Gly Gly Val Cys Trp Leu Gln GlnGly Gln Glu 35 40 45 Ala Thr Cys Ser Leu Val Leu Gln Thr Asp Val Thr ArgAla Glu 50 55 60 Cys Cys Ala Ser Gly Asn Ile Asp Thr Ala Trp Ser Asn LeuThr 65 70 75 His Pro Gly Asn Lys Ile Asn Leu Leu Gly Phe Leu Gly Leu Val80 85 90 His Cys Leu Pro Cys Lys Asp Ser Cys Asp Gly Val Glu Cys Gly 95100 105 Pro Gly Lys Ala Cys Arg Met Leu Gly Gly Arg Pro Arg Cys Glu 110115 120 Cys Ala Pro Asp Cys Ser Gly Leu Pro Ala Arg Leu Gln Val Cys 125130 135 Gly Ser Asp Gly Ala Thr Tyr Arg Asp Glu Cys Glu Leu Arg Ala 140145 150 Ala Arg Cys Arg Gly His Pro Asp Leu Ser Val Met Tyr Arg Gly 155160 165 Arg Cys Arg Lys Ser Cys Glu His Val Val Cys Pro Arg Pro Gln 170175 180 Ser Cys Val Val Asp Gln Thr Gly Ser Ala His Cys Val Val Cys 185190 195 Arg Ala Ala Pro Cys Pro Val Pro Ser Ser Pro Gly Gln Glu Leu 200205 210 Cys Gly Asn Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met Arg 215220 225 Gln Ala Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His Ala 230235 240 Gly Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly Gly Glu Ser 245250 255 Ala Glu Glu Glu Glu Asn Phe Val 260 5 2725 DNA Homo Sapien 5ggaggcggag gccgcggcga gccgggccga gcagtgaggg ccctagcggg 50 gcccgagcggggcccggggc ccctaagcca ttcctgaagt catgggctgg 100 ccaggacatt ggtgacccgccaatccggta tggacgactg gaagcccagc 150 cccctcatca agccctttgg ggctcggaagaagcggagct ggtaccttac 200 ctggaagtat aaactgacaa accagcgggc cctgcggagattctgtcaga 250 caggggccgt gcttttcctg ctggtgactg tcattgtcaa tatcaagttg300 atcctggaca ctcggcgagc catcagtgaa gccaatgaag acccagagcc 350agagcaagac tatgatgagg ccctaggccg cctggagccc ccacggcgca 400 gaggcagtggtccccggcgg gtcctggacg tagaggtgta ttcaagtcgc 450 agcaaagtat atgtggcagtggatggcacc acggtgctgg aggatgaggc 500 ccgggagcag ggccggggca tccatgtcattgtcctcaac caggccacgg 550 gccacgtgat ggcaaaacgt gtgtttgaca cgtactcacctcatgaggat 600 gaggccatgg tgctattcct caacatggta gcgcccggcc gagtgctcat650 ctgcactgtc aaggatgagg gctccttcca cctcaaggac acagccaagg 700ctctgctgag gagcctgggc agccaggctg gccctgccct gggctggagg 750 gacacatgggccttcgtggg acgaaaagga ggtcctgtct tcggggagaa 800 acattctaag tcacctgccctctcttcctg gggggaccca gtcctgctga 850 agacagatgt gccattgagc tcagcagaagaggcagagtg ccactgggca 900 gacacagagc tgaaccgtcg ccgccggcgc ttctgcagcaaagttgaggg 950 ctatggaagt gtatgcagct gcaaggaccc cacacccatc gagttcagcc1000 ctgacccact cccagacaac aaggtcctca atgtgcctgt ggctgtcatt 1050gcagggaacc gacccaatta cctgtacagg atgctgcgct ctctgctttc 1100 agcccagggggtgtctcctc agatgataac agttttcatt gacggctact 1150 atgaggaacc catggatgtggtggcactgt ttggtctgag gggcatccag 1200 catactccca tcagcatcaa gaatgcccgcgtgtctcagc actacaaggc 1250 cagcctcact gccactttca acctgtttcc ggaggccaagtttgctgtgg 1300 ttctggaaga ggacctggac attgctgtgg attttttcag tttcctgagc1350 caatccatcc acctactgga ggaggatgac agcctgtact gcatctctgc 1400ctggaatgac caggggtatg aacacacggc tgaggaccca gcactactgt 1450 accgtgtggagaccatgcct gggctgggct gggtgctcag gaggtccttg 1500 tacaaggagg agcttgagcccaagtggcct acaccggaaa agctctggga 1550 ttgggacatg tggatgcgga tgcctgaacaacgccggggc cgagagtgca 1600 tcatccctga cgtttcccga tcctaccact ttggcatcgtcggcctcaac 1650 atgaatggct actttcacga ggcctacttc aagaagcaca agttcaacac1700 ggttccaggt gtccagctca ggaatgtgga cagtctgaag aaagaagctt 1750atgaagtgga agttcacagg ctgctcagtg aggctgaggt tctggaccac 1800 agcaagaacccttgtgaaga ctctttcctg ccagacacag agggccacac 1850 ctacgtggcc tttattcgaatggagaaaga tgatgacttc accacctgga 1900 cccagcttgc caagtgcctc catatctgggacctggatgt gcgtggcaac 1950 catcggggcc tgtggagatt gtttcggaag aagaaccacttcctggtggt 2000 gggggtcccg gcttccccct actcagtgaa gaagccaccc tcagtcaccc2050 caattttcct ggagccaccc ccaaaggagg agggagcccc aggagcccca 2100gaacagacat gagacctcct ccaggaccct gcggggctgg gtactgtgta 2150 cccccaggctggctagccct tccctccatc ctgtaggatt ttgtagatgc 2200 tggtaggggc tggggctaccttgtttttaa catgagactt aattactaac 2250 tccaagggga gggttcccct gctccaacaccccgttcctg agttaaaagt 2300 ctatttattt acttccttgt tggagaaggg caggagagtacctgggaatc 2350 attacgatcc ctagcagctc atcctgccct ttgaataccc tcactttcca2400 ggcctggctc agaatctaac ctatttattg actgtcctga gggccttgaa 2450aacaggccga acctggaggg cctggatttc tttttgggct ggaatgctgc 2500 cctgagggtggggctggctc ttactcagga aactgctgtg cccaacccat 2550 ggacaggccc agctggggcccacatgctga cacagactca ctcagagacc 2600 cttagacact ggaccaggcc tcctctcagccttctctttg tccagatttc 2650 caaagctgga taagttggtc attgattaaa aaaggagaagccctctggga 2700 aaaaaaaaaa aaaaaaaaaa aaaaa 2725 6 660 PRT Homo Sapien 6Met Asp Asp Trp Lys Pro Ser Pro Leu Ile Lys Pro Phe Gly Ala 1 5 10 15Arg Lys Lys Arg Ser Trp Tyr Leu Thr Trp Lys Tyr Lys Leu Thr 20 25 30 AsnGln Arg Ala Leu Arg Arg Phe Cys Gln Thr Gly Ala Val Leu 35 40 45 Phe LeuLeu Val Thr Val Ile Val Asn Ile Lys Leu Ile Leu Asp 50 55 60 Thr Arg ArgAla Ile Ser Glu Ala Asn Glu Asp Pro Glu Pro Glu 65 70 75 Gln Asp Tyr AspGlu Ala Leu Gly Arg Leu Glu Pro Pro Arg Arg 80 85 90 Arg Gly Ser Gly ProArg Arg Val Leu Asp Val Glu Val Tyr Ser 95 100 105 Ser Arg Ser Lys ValTyr Val Ala Val Asp Gly Thr Thr Val Leu 110 115 120 Glu Asp Glu Ala ArgGlu Gln Gly Arg Gly Ile His Val Ile Val 125 130 135 Leu Asn Gln Ala ThrGly His Val Met Ala Lys Arg Val Phe Asp 140 145 150 Thr Tyr Ser Pro HisGlu Asp Glu Ala Met Val Leu Phe Leu Asn 155 160 165 Met Val Ala Pro GlyArg Val Leu Ile Cys Thr Val Lys Asp Glu 170 175 180 Gly Ser Phe His LeuLys Asp Thr Ala Lys Ala Leu Leu Arg Ser 185 190 195 Leu Gly Ser Gln AlaGly Pro Ala Leu Gly Trp Arg Asp Thr Trp 200 205 210 Ala Phe Val Gly ArgLys Gly Gly Pro Val Phe Gly Glu Lys His 215 220 225 Ser Lys Ser Pro AlaLeu Ser Ser Trp Gly Asp Pro Val Leu Leu 230 235 240 Lys Thr Asp Val ProLeu Ser Ser Ala Glu Glu Ala Glu Cys His 245 250 255 Trp Ala Asp Thr GluLeu Asn Arg Arg Arg Arg Arg Phe Cys Ser 260 265 270 Lys Val Glu Gly TyrGly Ser Val Cys Ser Cys Lys Asp Pro Thr 275 280 285 Pro Ile Glu Phe SerPro Asp Pro Leu Pro Asp Asn Lys Val Leu 290 295 300 Asn Val Pro Val AlaVal Ile Ala Gly Asn Arg Pro Asn Tyr Leu 305 310 315 Tyr Arg Met Leu ArgSer Leu Leu Ser Ala Gln Gly Val Ser Pro 320 325 330 Gln Met Ile Thr ValPhe Ile Asp Gly Tyr Tyr Glu Glu Pro Met 335 340 345 Asp Val Val Ala LeuPhe Gly Leu Arg Gly Ile Gln His Thr Pro 350 355 360 Ile Ser Ile Lys AsnAla Arg Val Ser Gln His Tyr Lys Ala Ser 365 370 375 Leu Thr Ala Thr PheAsn Leu Phe Pro Glu Ala Lys Phe Ala Val 380 385 390 Val Leu Glu Glu AspLeu Asp Ile Ala Val Asp Phe Phe Ser Phe 395 400 405 Leu Ser Gln Ser IleHis Leu Leu Glu Glu Asp Asp Ser Leu Tyr 410 415 420 Cys Ile Ser Ala TrpAsn Asp Gln Gly Tyr Glu His Thr Ala Glu 425 430 435 Asp Pro Ala Leu LeuTyr Arg Val Glu Thr Met Pro Gly Leu Gly 440 445 450 Trp Val Leu Arg ArgSer Leu Tyr Lys Glu Glu Leu Glu Pro Lys 455 460 465 Trp Pro Thr Pro GluLys Leu Trp Asp Trp Asp Met Trp Met Arg 470 475 480 Met Pro Glu Gln ArgArg Gly Arg Glu Cys Ile Ile Pro Asp Val 485 490 495 Ser Arg Ser Tyr HisPhe Gly Ile Val Gly Leu Asn Met Asn Gly 500 505 510 Tyr Phe His Glu AlaTyr Phe Lys Lys His Lys Phe Asn Thr Val 515 520 525 Pro Gly Val Gln LeuArg Asn Val Asp Ser Leu Lys Lys Glu Ala 530 535 540 Tyr Glu Val Glu ValHis Arg Leu Leu Ser Glu Ala Glu Val Leu 545 550 555 Asp His Ser Lys AsnPro Cys Glu Asp Ser Phe Leu Pro Asp Thr 560 565 570 Glu Gly His Thr TyrVal Ala Phe Ile Arg Met Glu Lys Asp Asp 575 580 585 Asp Phe Thr Thr TrpThr Gln Leu Ala Lys Cys Leu His Ile Trp 590 595 600 Asp Leu Asp Val ArgGly Asn His Arg Gly Leu Trp Arg Leu Phe 605 610 615 Arg Lys Lys Asn HisPhe Leu Val Val Gly Val Pro Ala Ser Pro 620 625 630 Tyr Ser Val Lys LysPro Pro Ser Val Thr Pro Ile Phe Leu Glu 635 640 645 Pro Pro Pro Lys GluGlu Gly Ala Pro Gly Ala Pro Glu Gln Thr 650 655 660 7 2395 DNA HomoSapien 7 cctggagccg gaagcgcggc tgcagcaggg cgaggctcca ggtggggtcg 50gttccgcatc cagcctagcg tgtccacgat gcggctgggc tccgggactt 100 tcgctacctgttgcgtagcg atcgaggtgc tagggatcgc ggtcttcctt 150 cggggattct tcccggctcccgttcgttcc tctgccagag cggaacacgg 200 agcggagccc ccagcgcccg aaccctcggctggagccagt tctaactgga 250 ccacgctgcc accacctctc ttcagtaaag ttgttattgttctgatagat 300 gccttgagag atgattttgt gtttgggtca aagggtgtga aatttatgcc350 ctacacaact taccttgtgg aaaaaggagc atctcacagt tttgtggctg 400aagcaaagcc acctacagtt actatgcctc gaatcaaggc attgatgacg 450 gggagccttcctggctttgt cgacgtcatc aggaacctca attctcctgc 500 actgctggaa gacagtgtgataagacaagc aaaagcagct ggaaaaagaa 550 tagtctttta tggagatgaa acctgggttaaattattccc aaagcatttt 600 gtggaatatg atggaacaac ctcatttttc gtgtcagattacacagaggt 650 ggataataat gtcacgaggc atttggataa agtattaaaa agaggagatt700 gggacatatt aatcctccac tacctggggc tggaccacat tggccacatt 750tcagggccca acagccccct gattgggcag aagctgagcg agatggacag 800 cgtgctgatgaagatccaca cctcactgca gtcgaaggag agagagacgc 850 ctttacccaa tttgctggttctttgtggtg accatggcat gtctgaaaca 900 ggaagtcacg gggcctcctc caccgaggaggtgaatacac ctctgatttt 950 aatcagttct gcgtttgaaa ggaaacccgg tgatatccgacatccaaagc 1000 acgtccaata gacggatgtg gctgcgacac tggcgatagc acttggctta1050 ccgattccaa aagacagtgt agggagcctc ctattcccag ttgtggaagg 1100aagaccaatg agagagcagt tgagattttt acatttgaat acagtgcagc 1150 ttagtaaactgttgcaagag aatgtgccgt catatgaaaa agatcctggg 1200 tttgagcagt ttaaaatgtcagaaagattg catgggaact ggatcagact 1250 gtacttggag gaaaagcatt cagaagtcctattcaacctg ggctccaagg 1300 ttctcaggca gtacctggat gctctgaaga cgctgagcttgtccctgagt 1350 gcacaagtgg cccagttctc accctgctcc tgctcagcgt cccacaggca1400 ctgcacagaa aggctgagct ggaagtccca ctgtcatctc ctgggttttc 1450tctgctcttt tatttggtga tcctggttct ttcggccgtt cacgtcattg 1500 tgtgcacctcagctgaaagt tcgtgctact tctgtggcct ctcgtggctg 1550 gcggcaggct gcctttcgtttaccagactc tggttgaaca cctggtgtgt 1600 gccaagtgct ggcagtgccc tggacagggggcctcaggga aggacgtgga 1650 gcagccttat cccaggcctc tgggtgtccc gacacaggtgttcacatctg 1700 tgctgtcagg tcagatgcct cagttcttgg aaagctaggt tcctgcgact1750 gttaccaagg tgattgtaaa gagctggcgg tcacagagga acaagccccc 1800cagctgaggg ggtgtgtgaa tcggacagcc tcccagcaga ggtgtgggag 1850 ctgcagctgagggaagaaga gacaatcggc ctggacactc aggagggtca 1900 aaaggagact tggtcgcaccactcatcctg ccacccccag aatgcatcct 1950 gcctcatcag gtccagattt ctttccaaggcggacgtttt ctgttggaat 2000 tcttagtcct tggcctcgga caccttcatt cgttagctggggagtggtgg 2050 tgaggcagtg aagaagaggc ggatggtcac actcagatcc acagagccca2100 ggatcaaggg acccactgca gtggcagcag gactgttggg cccccacccc 2150aaccctgcac agccctcatc ccctcttggc ttgagccgtc agaggccctg 2200 tgctgagtgtctgaccgaga cactcacagc tttgtcatca gggcacaggc 2250 ttcctcggag ccaggatgatctgtgccacg cttgcacctc gggcccatct 2300 gggctcatgc tctctctcct gctattgaattagtacctag ctgcacacag 2350 tatgtagtta ccaaaagaat aaacggcaat aattgagaaaaaaaa 2395 8 310 PRT Homo Sapien 8 Met Arg Leu Gly Ser Gly Thr Phe AlaThr Cys Cys Val Ala Ile 1 5 10 15 Glu Val Leu Gly Ile Ala Val Phe LeuArg Gly Phe Phe Pro Ala 20 25 30 Pro Val Arg Ser Ser Ala Arg Ala Glu HisGly Ala Glu Pro Pro 35 40 45 Ala Pro Glu Pro Ser Ala Gly Ala Ser Ser AsnTrp Thr Thr Leu 50 55 60 Pro Pro Pro Leu Phe Ser Lys Val Val Ile Val LeuIle Asp Ala 65 70 75 Leu Arg Asp Asp Phe Val Phe Gly Ser Lys Gly Val LysPhe Met 80 85 90 Pro Tyr Thr Thr Tyr Leu Val Glu Lys Gly Ala Ser His SerPhe 95 100 105 Val Ala Glu Ala Lys Pro Pro Thr Val Thr Met Pro Arg IleLys 110 115 120 Ala Leu Met Thr Gly Ser Leu Pro Gly Phe Val Asp Val IleArg 125 130 135 Asn Leu Asn Ser Pro Ala Leu Leu Glu Asp Ser Val Ile ArgGln 140 145 150 Ala Lys Ala Ala Gly Lys Arg Ile Val Phe Tyr Gly Asp GluThr 155 160 165 Trp Val Lys Leu Phe Pro Lys His Phe Val Glu Tyr Asp GlyThr 170 175 180 Thr Ser Phe Phe Val Ser Asp Tyr Thr Glu Val Asp Asn AsnVal 185 190 195 Thr Arg His Leu Asp Lys Val Leu Lys Arg Gly Asp Trp AspIle 200 205 210 Leu Ile Leu His Tyr Leu Gly Leu Asp His Ile Gly His IleSer 215 220 225 Gly Pro Asn Ser Pro Leu Ile Gly Gln Lys Leu Ser Glu MetAsp 230 235 240 Ser Val Leu Met Lys Ile His Thr Ser Leu Gln Ser Lys GluArg 245 250 255 Glu Thr Pro Leu Pro Asn Leu Leu Val Leu Cys Gly Asp HisGly 260 265 270 Met Ser Glu Thr Gly Ser His Gly Ala Ser Ser Thr Glu GluVal 275 280 285 Asn Thr Pro Leu Ile Leu Ile Ser Ser Ala Phe Glu Arg LysPro 290 295 300 Gly Asp Ile Arg His Pro Lys His Val Gln 305 310 9 3060DNA Homo Sapien 9 cgcgaggcgc ggggagcctg ggaccaggag cgagagccgc ctacctgcag50 ccgccgccca cggcacggca gccaccatgg cgctcctgct gtgcttcgtg 100 ctcctgtgcggagtagtgga tttcgccaga agtttgagta tcactactcc 150 tgaagagatg attgaaaaagccaaagggga aactgcctat ctgccatgca 200 aatttacgct tagtcccgaa gaccagggaccgctggacat cgagtggctg 250 atatcaccag ctgataatca gaaggtggat caagtgattattttatattc 300 tggagacaaa atttatgatg actactatcc agatctgaaa ggccgagtac350 attttacgag taatgatctc aaatctggtg atgcatcaat aaatgtaacg 400aatttacaac tgtcagatat tggcacatat cagtgcaaag tgaaaaaagc 450 tcctggtgttgcaaataaga agattcatct ggtagttctt gttaagcctt 500 caggtgcgag atgttacgttgatggatctg aagaaattgg aagtgacttt 550 aagataaaat gtgaaccaaa agaaggttcacttccattac agtatgagtg 600 gcaaaaattg tctgactcac agaaaatgcc cacttcatggttagcagaaa 650 tgacttcatc tgttatatct gtaaaaaatg cctcttctga gtactctggg700 acatacagct gtacagtcag aaacagagtg ggctctgatc agtgcctgtt 750gcgtctaaac gttgtccctc cttcaaataa agctggacta attgcaggag 800 ccattataggaactttgctt gctctagcgc tcattggtct tatcatcttt 850 tgctgtcgta aaaagcgcagagaagaaaaa tatgaaaagg aagttcatca 900 cgatatcagg gaagatgtgc cacctccaaagagccgtacg tccactgcca 950 gaagctacat cggcagtaat cattcatccc tggggtccatgtctccttcc 1000 aacatggaag gatattccaa gactcagtat aaccaagtac caagtgaaga1050 ctttgaacgc actcctcaga gtccgactct cccacctgct aagttcaagt 1100acccttacaa gactgatgga attacagttg tataaatatg gactactgaa 1150 gaatctgaagtattgtatta tttgacttta ttttaggcct ctagtaaaga 1200 cttaaatgtt ttttaaaaaaagcacaaggc acagagatta gagcagctgt 1250 aagaacacat ctactttatg caatggcattagacatgtaa gtcagatgtc 1300 atgtcaaaat tagtacgagc caaattcttt gttaaaaaaccctatgtata 1350 gtgacactga tagttaaaag atgttttatt atattttcaa taactaccac1400 taacaaattt ttaacttttc atatgcatat tctgatatgt ggtcttttag 1450gaaaagtatg gttaatagtt gatttttcaa aggaaatttt aaaattctta 1500 cgttctgtttaatgtttttg ctatttagtt aaatacattg aagggaaata 1550 cccgttcttt tccccttttatgcacacaac agaaacacgc gttgtcatgc 1600 ctcaaactat tttttatttg caactacatgatttcacaca attctcttaa 1650 acaacgacat aaaatagatt tccttgtata taaataacttacatacgctc 1700 cataaagtaa attctcaaag gtgctagaac aaatcgtcca cttctacagt1750 gttctcgtat ccaacagagt tgatgcacaa tatataaata ctcaagtcca 1800atattaaaaa cttaggcact tgactaactt taataaaatt tctcaaacta 1850 tatcaatatctaaagtgcat atatttttta agaaagatta ttctcaataa 1900 cttctataaa aataagtttgatggtttggc ccatctaact tcactactat 1950 tagtaagaac ttttaacttt taatgtgtagtaaggtttat tctacctttt 2000 tctcaacatg acaccaacac aatcaaaaac gaagttagtgaggtgctaac 2050 atgtgaggat taatccagtg attccggtca caatgcattc caggaggagg2100 tacccatgtc actggaattg ggcgatatgg tttatttttt cttccctgat 2150ttggataacc aaatggaaca ggaggaggat agtgattctg atggccattc 2200 cctcgatacattcctggctt ttttctgggc aaagggtgcc acattggaag 2250 aggtggaaat ataagttctgaaatctgtag ggaagagaac acattaagtt 2300 aattcaaagg aaaaaatcat catctatgttccagatttct cattaaagac 2350 aaagttaccc acaacactga gatcacatct aagtgacactcctattgtca 2400 ggtctaaata cattaaaaac ctcatgtgta ataggcgtat aatgtataac2450 aggtgaccaa tgttttctga atgcataaag aaatgaataa actcaaacac 2500agtacttcct aaacaacttc aaccaaaaaa gaccaaaaca tggaacgaat 2550 ggaagcttgtaaggacatgc ttgttttagt ccagtggttt ccacagctgg 2600 ctaagccagg agtcacttggaggcttttaa atacaaaaca ttggagctgg 2650 aggccattat ccttagcaaa ctaatgcagaaacagaaaat caactaccgc 2700 atgttctcac ttataagtgg gaggtaatga taagaacttatgaacacaaa 2750 gaaggaaaca atagacattg gagtctattt gagaggggag ggtgggagaa2800 ggaaaaggag cagaaaagat aactattgag tactgccttc acacctgggt 2850gatgaaataa tatgtacaac aaatccctgt gacacatgtt tacctatgga 2900 acaaaccttcatgtgtatcc ctaaacctaa aataaaagtt aaaaaaaaaa 2950 aaaraaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 3050 aaaaaaaaaa 3060 10 352 PRT Homo Sapien 10 MetAla Leu Leu Leu Cys Phe Val Leu Leu Cys Gly Val Val Asp 1 5 10 15 PheAla Arg Ser Leu Ser Ile Thr Thr Pro Glu Glu Met Ile Glu 20 25 30 Lys AlaLys Gly Glu Thr Ala Tyr Leu Pro Cys Lys Phe Thr Leu 35 40 45 Ser Pro GluAsp Gln Gly Pro Leu Asp Ile Glu Trp Leu Ile Ser 50 55 60 Pro Ala Asp AsnGln Lys Val Asp Gln Val Ile Ile Leu Tyr Ser 65 70 75 Gly Asp Lys Ile TyrAsp Asp Tyr Tyr Pro Asp Leu Lys Gly Arg 80 85 90 Val His Phe Thr Ser AsnAsp Leu Lys Ser Gly Asp Ala Ser Ile 95 100 105 Asn Val Thr Asn Leu GlnLeu Ser Asp Ile Gly Thr Tyr Gln Cys 110 115 120 Lys Val Lys Lys Ala ProGly Val Ala Asn Lys Lys Ile His Leu 125 130 135 Val Val Leu Val Lys ProSer Gly Ala Arg Cys Tyr Val Asp Gly 140 145 150 Ser Glu Glu Ile Gly SerAsp Phe Lys Ile Lys Cys Glu Pro Lys 155 160 165 Glu Gly Ser Leu Pro LeuGln Tyr Glu Trp Gln Lys Leu Ser Asp 170 175 180 Ser Gln Lys Met Pro ThrSer Trp Leu Ala Glu Met Thr Ser Ser 185 190 195 Val Ile Ser Val Lys AsnAla Ser Ser Glu Tyr Ser Gly Thr Tyr 200 205 210 Ser Cys Thr Val Arg AsnArg Val Gly Ser Asp Gln Cys Leu Leu 215 220 225 Arg Leu Asn Val Val ProPro Ser Asn Lys Ala Gly Leu Ile Ala 230 235 240 Gly Ala Ile Ile Gly ThrLeu Leu Ala Leu Ala Leu Ile Gly Leu 245 250 255 Ile Ile Phe Cys Cys ArgLys Lys Arg Arg Glu Glu Lys Tyr Glu 260 265 270 Lys Glu Val His His AspIle Arg Glu Asp Val Pro Pro Pro Lys 275 280 285 Ser Arg Thr Ser Thr AlaArg Ser Tyr Ile Gly Ser Asn His Ser 290 295 300 Ser Leu Gly Ser Met SerPro Ser Asn Met Glu Gly Tyr Ser Lys 305 310 315 Thr Gln Tyr Asn Gln ValPro Ser Glu Asp Phe Glu Arg Thr Pro 320 325 330 Gln Ser Pro Thr Leu ProPro Ala Lys Phe Lys Tyr Pro Tyr Lys 335 340 345 Thr Asp Gly Ile Thr ValVal 350 11 983 DNA Homo Sapien 11 ggatgcagca gagaggagca gctggaagccgtggctgcgc tctcttccct 50 ctgctgggcg tcctgttctt ccagggtgtt tatatcgtcttttccttgga 100 gattcgtgca gatgcccatg tccgaggtta tgttggagaa aagatcaagt150 tgaaatgcac tttcaagtca acttcagatg tcactgacaa gcttactata 200gactggacat atcgccctcc cagcagcagc cacacagtat caatatttca 250 ttatcagtctttccagtacc caaccacagc aggcacattt cgggatcgga 300 tttcctgggt tggaaatgtatacaaagggg atgcatctat aagtataagc 350 aaccctacca taaaggacaa tgggacattcagctgtgctg tgaagaatcc 400 cccagatgtg caccataata ttcccatgac agagctaacagtcacagaaa 450 ggggttttgg caccatgctt tcctctgtgg cccttctttc catccttgtc500 tttgtgccct cagccgtggt ggttgctctg ctgctggtga gaatggggag 550gaaggctgct gggctgaaga agaggagcag gtctggctat aagaagtcat 600 ctattgaggtttccgatgac actgatcagg aggaggaaga ggcgtgtatg 650 gcgaggcttt gtgtccgttgcgctgagtgc ctggattcag actatgaaga 700 gacatattga tgaaagtctg tatgacacaagaagagtcac ctaaagacag 750 gaaacatccc attccactgg cagctaaagc ctgtcagagaaagtggagct 800 ggcctggacc atagcgatgg acaatcctgg agatcatcag taaagacttt850 aggaaccact tatttattga ataaatgttc ttgttgtatt tataaactgt 900tcaggaagtc tcataagaga ctcatgactt cccctttcaa tgaattatgc 950 tgtaattgaatgaagaaatt cttttcctga gca 983 12 235 PRT Homo Sapien 12 Met Gln Gln ArgGly Ala Ala Gly Ser Arg Gly Cys Ala Leu Phe 1 5 10 15 Pro Leu Leu GlyVal Leu Phe Phe Gln Gly Val Tyr Ile Val Phe 20 25 30 Ser Leu Glu Ile ArgAla Asp Ala His Val Arg Gly Tyr Val Gly 35 40 45 Glu Lys Ile Lys Leu LysCys Thr Phe Lys Ser Thr Ser Asp Val 50 55 60 Thr Asp Lys Leu Thr Ile AspTrp Thr Tyr Arg Pro Pro Ser Ser 65 70 75 Ser His Thr Val Ser Ile Phe HisTyr Gln Ser Phe Gln Tyr Pro 80 85 90 Thr Thr Ala Gly Thr Phe Arg Asp ArgIle Ser Trp Val Gly Asn 95 100 105 Val Tyr Lys Gly Asp Ala Ser Ile SerIle Ser Asn Pro Thr Ile 110 115 120 Lys Asp Asn Gly Thr Phe Ser Cys AlaVal Lys Asn Pro Pro Asp 125 130 135 Val His His Asn Ile Pro Met Thr GluLeu Thr Val Thr Glu Arg 140 145 150 Gly Phe Gly Thr Met Leu Ser Ser ValAla Leu Leu Ser Ile Leu 155 160 165 Val Phe Val Pro Ser Ala Val Val ValAla Leu Leu Leu Val Arg 170 175 180 Met Gly Arg Lys Ala Ala Gly Leu LysLys Arg Ser Arg Ser Gly 185 190 195 Tyr Lys Lys Ser Ser Ile Glu Val SerAsp Asp Thr Asp Gln Glu 200 205 210 Glu Glu Glu Ala Cys Met Ala Arg LeuCys Val Arg Cys Ala Glu 215 220 225 Cys Leu Asp Ser Asp Tyr Glu Glu ThrTyr 230 235 13 924 DNA Homo Sapien 13 aaggagcagc ccgcaagcac caagtgagaggcatgaagtt acagtgtgtt 50 tccctttggc tcctgggtac aatactgata ttgtgctcagtagacaacca 100 cggtctcagg agatgtctga tttccacaga catgcaccat atagaagaga150 gtttccaaga aatcaaaaga gccatccaag ctaaggacac cttcccaaat 200gtcactatcc tgtccacatt ggagactctg cagatcatta agcccttaga 250 tgtgtgctgcgtgaccaaga acctcctggc gttctacgtg gacagggtgt 300 tcaaggatca tcaggagccaaaccccaaaa tcttgagaaa aatcagcagc 350 attgccaact ctttcctcta catgcagaaaactctgcggc aatgtcagga 400 acagaggcag tgtcactgca ggcaggaagc caccaatgccaccagagtca 450 tccatgacaa ctatgatcag ctggaggtcc acgctgctgc cattaaatcc500 ctgggagagc tcgacgtctt tctagcctgg attaataaga atcatgaagt 550aatgttctca gcttgatgac aaggaacctg tatagtgatc cagggatgaa 600 caccccctgtgcggtttact gtgggagaca gcccaccttg aaggggaagg 650 agatggggaa ggccccttgcagctgaaagt cccactggct ggcctcaggc 700 tgtcttattc cgcttgaaaa taggcaaaaagtctactgtg gtatttgtaa 750 taaactctat ctgctgaaag ggcctgcagg ccatcctgggagtaaagggc 800 tgccttccca tctaatttat tgtaaagtca tatagtccat gtctgtgatg850 tgagccaagt gatatcctgt agtacacatt gtactgagtg gtttttctga 900ataaattcca tattttacct atga 924 14 177 PRT Homo Sapien 14 Met Lys Leu GlnCys Val Ser Leu Trp Leu Leu Gly Thr Ile Leu 1 5 10 15 Ile Leu Cys SerVal Asp Asn His Gly Leu Arg Arg Cys Leu Ile 20 25 30 Ser Thr Asp Met HisHis Ile Glu Glu Ser Phe Gln Glu Ile Lys 35 40 45 Arg Ala Ile Gln Ala LysAsp Thr Phe Pro Asn Val Thr Ile Leu 50 55 60 Ser Thr Leu Glu Thr Leu GlnIle Ile Lys Pro Leu Asp Val Cys 65 70 75 Cys Val Thr Lys Asn Leu Leu AlaPhe Tyr Val Asp Arg Val Phe 80 85 90 Lys Asp His Gln Glu Pro Asn Pro LysIle Leu Arg Lys Ile Ser 95 100 105 Ser Ile Ala Asn Ser Phe Leu Tyr MetGln Lys Thr Leu Arg Gln 110 115 120 Cys Gln Glu Gln Arg Gln Cys His CysArg Gln Glu Ala Thr Asn 125 130 135 Ala Thr Arg Val Ile His Asp Asn TyrAsp Gln Leu Glu Val His 140 145 150 Ala Ala Ala Ile Lys Ser Leu Gly GluLeu Asp Val Phe Leu Ala 155 160 165 Trp Ile Asn Lys Asn His Glu Val MetPhe Ser Ala 170 175

What is claimed:
 1. Isolated nucleic acid having at least 80% nucleicacid sequence identity to: (a) a nucleotide sequence encoding thepolypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6(SEQ ID NO:6), FIG. 8 (SEQ ID NO:8), FIG. 10 (SEQ ID NO: 10), FIG. 12(SEQ ID NO:12) or FIG. 14 (SEQ ID NO:14); (b) a nucleotide sequenceencoding the polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQ ID NO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQ ID NO:14), lacking itsassociated signal peptide; (c) a nucleotide sequence encoding anextracellular domain of the polypeptide shown in FIG. 2 (SEQ ID NO:2),FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQ ID NO:8), FIG.10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQ ID NO:14),with its associated signal peptide; or (d) a nucleotide sequenceencoding an extracellular domain of the polypeptide shown in FIG. 2 (SEQID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQ IDNO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQ IDNO:14), lacking its associated signal peptide.
 2. Isolated nucleic acidhaving at least 80% nucleic acid sequence identity to a nucleotidesequence selected from the group consisting of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3), FIG. 5 (SEQ IDNO:5), FIG. 7 (SEQ ID NO:7), FIG. 9 (SEQ ID NO:9), FIG. 11 (SEQ IDNO:11) and FIG. 13 (SEQ ID NO:13).
 3. Isolated nucleic acid having atleast 80% nucleic acid sequence identity to a nucleotide sequenceselected from the group consisting of the full-length coding sequence ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ IDNO:3), FIG. 5 (SEQ ID NO:5), FIG. 7 (SEQ ID NO:7), FIG. 9 (SEQ ID NO:9),FIG. 11 (SEQ ID NO:11) and FIG. 13 (SEQ ID NO:13).
 4. Isolated nucleicacid having at least 80% nucleic acid sequence identity to thefull-length coding sequence of the DNA deposited under any ATCCaccession number shown in Table
 7. 5. A vector comprising the nucleicacid of claim
 1. 6. The vector of claim 5 operably linked to controlsequences recognized by a host cell transformed with the vector.
 7. Ahost cell comprising the vector of claim
 5. 8. The host cell of claim 7,wherein said cell is a CHO cell, an E. coli cell or a yeast cell.
 9. Aprocess for producing a PRO polypeptide comprising culturing the hostcell of claim 7 under conditions suitable for expression of said PROpolypeptide and recovering said PRO polypeptide from the cell culture.10. An isolated polypeptide having at least 80% amino acid sequenceidentity to: (a) an amino acid sequence of the polypeptide shown in FIG.2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQID NO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQID NO:14); (b) an amino acid sequence of the polypeptide shown in FIG. 2(SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQID NO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQID NO:14), lacking its associated signal peptide; (c) an amino acidsequence of an extracellular domain of the polypeptide shown in FIG. 2(SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQID NO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQID NO:14), with its associated signal peptide; or (d) an amino acidsequence of an extracellular domain of the polypeptide shown in FIG. 2(SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6), FIG. 8 (SEQID NO:8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO:12) or FIG. 14 (SEQID NO:14), lacking its associated signal peptide.
 11. An isolatedpolypeptide having at least 80% amino acid sequence identity to an aminoacid sequence encoded by the full-length coding sequence of the DNAdeposited under any ATCC accession number shown in Table
 7. 12. Achimeric molecule comprising a polypeptide according to claim 10 fusedto a heterologous amino acid sequence.
 13. The chimeric molecule ofclaim 12, wherein said heterologous amino acid sequence is an epitopetag sequence or an Fc region of an immunoglobulin.
 14. An antibody whichspecifically binds to a polypeptide according to claim
 10. 15. Theantibody of claim 14, wherein said antibody is a monoclonal antibody, ahumanized antibody or a single-chain antibody.
 16. A composition ofmatter comprising (a) a polypeptide of claim 10, (b) an agonist of saidpolypeptide, (c) an antagonist of said polypeptide, or (d) an antibodythat binds to said polypeptide, in combination with a carrier.
 17. Thecomposition of matter of claim 16, wherein said carrier is apharmaceutically acceptable carrier.
 18. The composition of matter ofclaim 16 which is useful for the treatment of an immune related diseasein a mammal.
 19. The composition of matter of claim 16, wherein (a),(b), (c) or (d) is capable of (i) enhancing the proliferation ofT-lymphocytes in a mammal, (ii) inhibiting the proliferation ofT-lymphocytes in a mammal, or (iii) increasing infiltration ofinflammatory cells into a tissue of a mammal.
 20. The composition ofmatter of claim 16 comprising a therapeutically effective amount of (a),(b), (c) or (d).
 21. An article of manufacture, comprising: a container;a label on said container; and a composition of matter comprising (a) apolypeptide of claim 10, (b) an agonist of said polypeptide, (c) anantagonist of said polypeptide, or (d) an antibody that binds to saidpolypeptide, contained within said container, wherein label on saidcontainer indicates that said composition of matter can be used fortreating an immune related disease.
 22. A method of treating an immunerelated disorder in a mammal in need thereof comprising administering tosaid mammal a therapeutically effective amount of (a) a polypeptide ofclaim 10, (b) an agonist of said polypeptide, (c) an antagonist of saidpolypeptide, or (d) an antibody that binds to said polypeptide.
 23. Themethod of claim 22, wherein the immune related disorder is systemiclupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenilechronic arthritis, a spondyloarthropathy, systemic sclerosis, anidiopathic inflammatory myopathy, Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renaldisease, a demyelinating disease of the central or peripheral nervoussystem, idiopathic demyelinating polyneuropathy, Guillain-Barresyndrome, a chronic inflammatory demyelinating polyneuropathy, ahepatobiliary disease, infectious or autoimmune chronic activehepatitis, primary biliary cirrhosis, granulomatous hepatitis,sclerosing cholangitis, inflammatory bowel disease, gluten-sensitiveenteropathy, Whipple's disease, an autoimmune or immune-mediated skindisease, a bullous skin disease, erythema multiforme, contactdermatitis, psoriasis, an allergic disease, asthma, allergic rhinitis,atopic dermatitis, food hypersensitivity, urticaria, an immunologicdisease of the lung, eosinophilic pneumonias, idiopathic pulmonaryfibrosis, hypersensitivity pneumonitis, a transplantation associateddisease, graft rejection or graft-versus-host-disease.
 24. A method fordetermining the presence of a PRO 1265, PRO 1 308, PRO 1475, PRO4405,PRO5723, PRO7425 or PRO9940 polypeptide in a sample suspected ofcontaining said polypeptide, said method comprising exposing said sampleto an anti-PRO1265, anti-PRO1308, anti-PRO1475, anti-PRO4405,anti-PRO5723, anti-PRO7425 or anti-PRO9940 antibody and determiningbinding of said antibody to a component of said sample.
 25. A method ofdiagnosing an immune related disease in a mammal, said method comprisingdetecting the level of expression of a gene encoding a PRO1265, PRO1308,PRO1475, PRO4405, PRO5723, PRO7425 or PRO9940 polypeptide (a) in a testsample of tissue cells obtained from the mammal, and (b) in a controlsample of known normal tissue cells of the same cell type, wherein ahigher or lower level of expression of said gene in the test sample ascompared to the control sample is indicative of the presence of animmune related disease in the mammal from which the test tissue cellswere obtained.
 26. A method of diagnosing an immune related disease in amammal, said method comprising (a) contacting an anti-PRO1265,anti-PRO1308, anti-PRO 1475, anti-PRO4405, anti-PRO5723, anti-PRO7425 oranti-PRO9940 antibody with a test sample of tissue cells obtained fromsaid mammal and (b) detecting the formation of a complex between theantibody and the polypeptide in the test sample, wherein formation ofsaid complex is indicative of the presence of an immune related diseasein the mammal from which the test tissue cells were obtained.
 27. Amethod of identifying a compound that inhibits the activity of a PRO1265, PRO 1308, PRO1475, PRO4405, PRO5723, PRO7425 or PRO9940polypeptide, said method comprising contacting cells which normallyrespond to said polypeptide with (a) said polypeptide and (b) acandidate compound, and determining the lack responsiveness by said cellto (a).
 28. A method of identifying a compound that inhibits theexpression of a gene encoding a PRO 1265, PRO 1308, PRO 1475, PRO4405,PRO5723, PRO7425 or PRO9940 polypeptide, said method comprisingcontacting cells which normally express said polypeptide with acandidate compound, and determining the lack of expression said gene.29. The method of claim 28, wherein said candidate compound is anantisense nucleic acid.
 30. A method of identifying a compound thatmimics the activity of a PRO1265, PRO1308, PRO 1475, PRO4405, PRO5723,PRO7425 or PRO9940 polypeptide, said method comprising contacting cellswhich normally respond to said polypeptide with a candidate compound,and determining the responsiveness by said cell to said candidatecompound.
 31. A method of stimulating the proliferation ofT-lymphocytes, said method comprising contacting T-lymphocytes with aneffective amount of (a) a PRO 1475, PRO5723, PRO7425 or PRO9940polypeptide or (b) an agonist of (a).
 32. A method of inhibiting theproliferation of T-lymphocytes, said method comprising contactingT-lymphocytes with an effective amount of an antagonist of a PRO 1475,PRO5723, PRO7425 or PRO9940 polypeptide.
 33. A method of inhibiting theproliferation of T-lymphocytes, said method comprising contactingT-lymphocytes with an effective amount of (a) a PRO 1265, PRO 1308 orPRO4405 polypeptide or (b) an agonist of (a).
 34. A method ofstimulating the proliferation of T-lymphocytes, said method comprisingcontacting T-lymphocytes with an effective amount of an antagonist of aPRO 1265, PRO 1308 or PRO4405 polypeptide.